Multi-focused laboratory experiments based on Quorum Sensing and Quorum Quenching for acquiring Microbial Physiology concepts.

After a time away from the classrooms and laboratories due to the global pandemic, the return to teaching activities during the semester represented a challenge to both teachers and students. Our particular situation in a Microbial Physiology course was the necessity of imparting in shorter time, laboratory practices that usually take longer. This article describes a 2-week-long laboratory exercise that covers several concepts in an interrelated way: conjugation as a gene transfer mechanism, regulation of microbial physiology, production of secondary metabolites, degradation of macromolecules, and biofilm formation. Utilizing a Quorum Quenching (QQ) strategy, the Quorum Sensing (QS) system of Pseudomonas aeruginosa is first attenuated. Then, phenotypes regulated by QS are evidenced. QS is a regulatory mechanism of microbial physiology that relies on signal molecules. QS is related in P. aeruginosa to several virulence factors, some of which are exploited in the laboratory practices presented in this work. QQ is a phenomenon by which QS is interrupted or attenuated. We utilized a QQ approach based on the enzymatic degradation of the P. aeruginosa QS signals to evidence QS-regulated traits that are relevant to our Microbial Physiology course. Results obtained with the same test performed by a random group of students before and after the activities show the positive effectiveness of the approach presented in this work.

Vinasse as a substrate for inoculant culture and soil fertigation: Advancing the circular and green economy.

Vinasse is a by-product with a key role in circular economy. In this work, we analyze sugarcane vinasse as culture medium for obtaining single and mixed inoculants. Trichoderma harzianum MT2 was cultured in single and sequential co-culture with Pseudomonas capeferrum WCS358 or Rhizobium sp. N21.2. Fungal biomass in single culture was more than three folds higher in vinasse than in a standard medium, and was higher in co-culture with Rhizobium sp. N21.2 than with P. capeferrum WCS358. Bacterial growths in vinasse, in particular P. capeferrum WCS358, were improved in co-culture with T. harzianum MT2. Residual vinasses, obtained after microbial growth, presented almost neutral pH and lower conductivities and toxicity than raw vinasse. Fertigation with residual vinasses modifies characteristics of soil evidenced in the total N, cation exchange capacity, urease and acid phosphatase, and microbial metabolic diversity, in comparison to raw vinasse. In general, soil fertigation with residual vinasse from co-culture with P. capeferrum WCS358 is more similar to irrigation with water. Treatment evaluation indicates that vinasse is suitable for the production of mixed inoculants containing T. harzianum. The co-culture with P. capeferrum WCS358 improves the characteristics of the residual vinasse allowing a fertigation with less detrimental effect in soil in comparison to Rhizobium sp. N21.2. Obtaining valuable biomass of single or mixed inoculants in vinasse with lower ecological impact is relevant for the circular and green economy.

Ivermectin compared with placebo in the clinical course in Mexican patients with asymptomatic and mild COVID-19: a randomized clinical trial.

BACKGROUND: Despite the development and application of vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) around the world, the scientific community is still trying to find some therapies to avoid or ameliorate the fatal evolution of the Coronavirus disease 2019 (COVID-19). Since the publication of the potential use of ivermectin as a treatment against the disease, a pleiad of information about it has been published. However, the evidence is not strong or weak enough to conclude its usefulness in the clinical evolution of patients infected with SARS-CoV-2. We evaluate the efficacy and safety of ivermectin in the treatment of Mexican patients with asymptomatic and mild COVID-19 in a three-day administration in comparison to placebo. METHODS: A randomized, double-blind, placebo-controlled trial was carried out in 66 adults with asymptomatic and mild COVID-19. Patients were randomly assigned 1:1 ratio to ivermectin plus acetaminophen or placebo plus acetaminophen. The primary endpoint was the proportion of subjects without a disease progression to severity according to COVID-19 guidelines by the National Institutes of Health (NIH) since randomization to 14 days. RESULTS: None of the participants presented progression to a severe state in either group. Viral load was measured on Days 1, 5, and 14. No significant differences were observed in baseline or 14-day between groups (p = 0.720 and 0.362, respectively). However, on Day 5, a significant difference in viral load was observed between groups (p = 0.039). The frequency of symptoms was similar between groups, and no significant differences were observed. The most frequent symptom was cough. One severe adverse event associated with SARS-CoV-2 infection was observed in the ivermectin group. CONCLUSIONS: At standard doses, ivermectin is not effective to prevent progression to a severe state or reducing symptoms in adults with asymptomatic and mild COVID-19. Trial registration The study was registered with ClinicalTrial.gov (NCT04407507) on May 29, 2020.

Characterization of the B-Cell Epitopes of Echinococcus granulosus Histones H4 and H2A Recognized by Sera From Patients With Liver Cysts.

Cystic echinococcosis (CE) is a zoonotic disease worldwide distributed, caused by the cestode Echinococcus granulosus sensu lato (E. granulosus), with an incidence rate of 50/100,000 person/year and a high prevalence in humans of 5-10%. Serology has variable sensitivity and specificity and low predictive values. Antigens used are from the hydatid fluid and recombinant antigens have not demonstrated superiority over hydatid fluid. A cell line called EGPE was obtained from E. granulosus sensu lato G1 strain from bovine liver. Serum from CE patients recognizes protein extracts from EGPE cells with higher sensitivity than protein extracts from hydatid fluid. In the present study, EGPE cell protein extracts and supernatants from cell colonies were eluted from a protein G affinity column performed with sera from 11 CE patients. LC-MS/MS proteomic analysis of the eluted proteins identified four E. granulosus histones: one histone H4 in the cell extract and supernatant, one histone H2A only in the cell extract, and two histones H2A only in the supernatant. This differential distribution of histones could reflect different parasite viability stages regarding their role in gene transcription and silencing and could interact with host cells. Bioinformatics tools characterized the linear and conformational epitopes involved in antibody recognition. The three-dimensional structure of each histone was obtained by molecular modeling and validated by molecular dynamics simulation and PCR confirmed the presence of the epitopes in the parasite genome. The three histones H2A were very different and had a less conserved sequence than the histone H4. Comparison of the histones of E. granulosus with those of other organisms showed exclusive regions for E. granulosus. Since histones play a role in the host-parasite relationship they could be good candidates to improve the predictive value of serology in CE.

In Search of a Neurotologic Profile in COVID-19 - A Study in Health Care Workers.

Introduction COVID-19 is an emerging disease and the neurotologic symptoms are still not well understood. Furthermore, the development of a neurotological profile and its associated factors can help the clinician in the diagnosis and treatment of this disease. The objective is to determine the neurotologic manifestations experienced by COVID-19 positive health care workers and their associated factors. Methods A symptoms survey was administered to health care workers who were positive to COVID-19 from September to October 2020. An informed consent form was digitally signed and Google Forms software was used for the survey. Frequencies and percentages were used for categorical variables, and associated clinical features were reported with odds ratios. Results We included 209 COVID-19 positive health care workers, 55.5% (n = 116) were women, and 44.5% (n = 93) were men. Fifty-three percent of patients were 20 to 30 years old and 56.4% had at least one comorbidity. The prevalence of neurotological manifestations was 18.6% (n = 39/209), the most frequent symptoms were vertigo (61.5%, n = 24/39), tinnitus (43.5%, n = 17/39), imbalance (43.5%, n = 17/39), and one case of facial paralysis (2.5%, n = 1/39). Neurotological manifestations were associated predominantly with asthenia (p = 0.021), loss of smell (p = 0.002) and taste dysfunction (p = 0.002). Conclusion The most common neurotological manifestations were vertigo, tinnitus and imbalance. Clinical features associated with a neurotologic profile were asthenia, hyposmia and dysgeusia.

Preliminary Study on Citrus Oils Antibacterial Activity Measured by Flow Cytometry: A Step-by-Step Development.

Essential oils of Citrus sinensis and Citrus latifolia have shown biological functions as antiseptics, anti-inflammatories, antioxidants, antifungal and antimutagenic, so the evaluation of their antibacterial capacity, by themselves or in combination with standard antibiotics, presents an alternative for infection treatment. Flow cytometry opens the door for the design of faster and more accurate measurement of antibacterial activity. We use a SYTO9/PI staining system on E. coli ATCC 25922 to determine antibacterial activity by counting live and dead cells through flow cytometry. We found that dual staining showed highly variable results due to wavelength overlapping and instead we used fluorochrome individual staining that highly correlated with viable counts. Chloramphenicol and cefotaxime treatments did not present a dose-response behavior, rendered diffuse readings and/or gave filament formation on fluorescence microscopy. Amikacin was a better comparison standard because it presented a dose-response behavior. Essential oils had low antibacterial activity as compared to amikacin, with a maximum of 10% and 20% for C. latifolia and C. sinensis, respectively. Combinations of essential oils with antibiotic resulted in an unforeseen strong inhibition of amikacin activity. Although a low antibacterial activity was found, a series of standardization steps are proposed for antibacterial activity measurement by flow cytometry.

Cost-effective optimized scleroglucan production by Sclerotium rolfsii ATCC 201126 at bioreactor scale. A quantity-quality assessment.

Exopolysaccharide (EPS) secretion by Sclerotium rolfsii ATCC 201126 in submerged cultures, already identified as high-osmolarity responsive, was assessed by reducing C-source without compromising EPS yields. A designed medium with 80 g sucrose L-1 (MOPT80) was tested at 3 L-bioreactor scale at different temperature, agitation, aeration and pH (uncontrolled vs. controlled) values. Optimal operative conditions (200 rpm, 28 °C, 0.5 vvm and initial pH -pHi- 4.5) were validated, as well as the possibility to work at pHi 5.5 to reduce biomass production. Purified EPSs produced in MOPT80 at optimal and other valid operative conditions exhibited refined grade (<1 % proteins and ash, 3-4 % reducing sugars, 87-99 % total sugars). EPS purity, MW and rheological parameters led to discourage pH controlled at 4.5. Relatively constant MW (6-8 × 106 Da) and outstanding viscosifying ability were found. Polyphasic EPS analysis (titre, purity, macromolecular features and rheological fitness) would support to properly select production conditions.

Characterization of BIP protein of G. lamblia as a potential immunogen in a mouse infection model.

Giardia lamblia is a protozoan parasite that causes one of the most common gastrointestinal diseases worldwide. To eliminate the parasite from the host intestine, it is necessary the activation of B-cell and T-cell dependent mechanisms. The knowledge about Giardia antigens that can stimulate the host immune response is limited. Recently, it has been described the Binding Immunoglobulin Protein (BIP) of G. lamblia (71kDa) as a potential immunogen. Additionally, our group has identified a highly immunogenic antigen (5G8 protein) of G. lamblia with a relative molecular mass of approximately 70kDa. There is some evidence suggesting that the 5G8 protein may activate both humoral and cellular immune responses. Based on these observations and preliminary mass spectrometry analyses, we hypothesized that the antigen 5G8 could be the BIP protein. In the present study, we characterize immunochemically the BIP protein of Giardia. Flow cytometric assays and western blotting were used to determine the expression profile of BIP and 5G8 antigens in Giardia trophozoites. The differences in expression profile indicated that BIP and 5G8 are not the same molecule. ELISA and Western blotting assays revealed that BIP protein was recognized by antibodies produced during G. lamblia infection in C3H/HeN mice. MTT assays did not reveal the activation of cellular immune response induced by BIP protein in vitro. In addition, we identified the potential B-cell and T-cell epitopes of G. lamblia BIP protein. This molecule is a conserved protein among Giardia strains and other pathogens. The complete immunological characterization of this antigen will contribute to a better understanding of the host-parasite interactions in Giardia infection.

Isolation and partial characterization of an immunogenic antigen of Giardia lamblia.

Humoral and cellular immune responses play an important role during Giardia lamblia infection. Several Giardia proteins have been identified as immunogenic antigens based on their elicited humoral immune response. Poorly is known about Giardia antigens that stimulate a cellular immune response. The main purpose of this study was to isolate and partial characterize an immunogenic antigen (5G8) of G. lamblia. The 5G8 protein was isolated from G. lamblia trophozoite lysates by affinity chromatography using moAb 5G8-coupled CNBr-Sepharose. The isolated protein was analysed by electrospray tandem mass spectrometry (ESI-MS/MS), and by diverse bioinformatics tools (GiardiaDB, BLASTn, BLASTp and ExPASy). Additionally, several biochemical and immunological characteristics of the isolated protein were analysed. By ESI-MS/MS the amino acidic 5G8 sequence was deduced. The 5G8 antigen belongs to the VSP family proteins of G. lamblia. This protein is composed by one polypeptide chain (±71kDa). Using the algorithm SYFPHEITI, we identified candidate CD4+ T-cell epitopes from the 5G8 antigen, which can elicit cell-mediated immune responses. In this study, we have identified a G. lamblia protein that induces a strong immune response in infected mice. The biochemical and immunological characterization of the immunogenic 5G8 antigen may contribute to the rational design of a Giardia vaccine.

Microbial production of scleroglucan and downstream processing.

Synthetic petroleum-based polymers and natural plant polymers have the disadvantage of restricted sources, in addition to the non-biodegradability of the former ones. In contrast, eco-sustainable microbial polysaccharides, of low-cost and standardized production, represent an alternative to address this situation. With a strong global market, they attracted worldwide attention because of their novel and unique physico-chemical properties as well as varied industrial applications, and many of them are promptly becoming economically competitive. Scleroglucan, a ß-1,3-ß-1,6-glucan secreted by Sclerotium fungi, exhibits high potential for commercialization and may show different branching frequency, side-chain length, and/or molecular weight depending on the producing strain or culture conditions. Water-solubility, viscosifying ability and wide stability over temperature, pH and salinity make scleroglucan useful for different biotechnological (enhanced oil recovery, food additives, drug delivery, cosmetic and pharmaceutical products, biocompatible materials, etc.), and biomedical (immunoceutical, antitumor, etc.) applications. It can be copiously produced at bioreactor scale under standardized conditions, where a high exopolysaccharide concentration normally governs the process optimization. Operative and nutritional conditions, as well as the incidence of scleroglucan downstream processing will be discussed in this chapter. The relevance of using standardized inocula from selected strains and experiences concerning the intricate scleroglucan scaling-up will be also herein outlined.

Purification and characterization of the membrane-bound quinoprotein glucose dehydrogenase of Gluconacetobacter diazotrophicus PAL 5.

Acetic acid bacteria oxidize a great number of substrates, such as alcohols and sugars, using different enzymes that are anchored to the membrane. In particular, Gluconacetobacter diazotrophicus is distinguished for its N2-fixing activity under high-aeration conditions. Ga. diazotrophicus is a true endophyte that also has membrane-bound enzymes to oxidize sugars and alcohols. Here we reported the purification and characterization of the membrane-bound glucose dehydrogenase (GDHm), an oxidoreductase of Ga. diazotrophicus. GDHm was solubilized and purified by chromatographic methods. Purified GDHm was monomeric, with a molecular mass of 86 kDa. We identified the prosthetic group as pyrroloquinoline quinone, whose redox state was reduced. GDHm showed an optimum pH of 7.2, and its isoelectric point was 6.0. This enzyme preferentially oxidized D-glucose, 2-deoxy-D-glucose, D-galactose and D-xylose; its affinity towards glucose was ten times greater than that of E. coli GDHm. Finally, Ga. diazotrophicus GDHm was capable of reducing quinones such as Q 1, Q 2, and decylubiquinone; this activity was entirely abolished in the presence of micromolar concentrations of the inhibitor, myxothiazol. Hence, our purification method yielded a highly purified GDHm whose molecular and kinetic parameters were determined. The possible implications of GDHm activity in the mechanism for reducing competitor microorganisms, as well as its participation in the respiratory system of Ga. diazotrophicus, are discussed.

Survey of Mexican retail stores for US beef product.

Retail packages (N=1004) containing fresh US beef in display cases in five cities across three regions of Mexico were surveyed for cut types, cutting styles, fat thickness measurements, marbling scores, and USDA Quality Grades to gain an overview of fresh US beef in Mexican retail markets. Data were analyzed to generate frequency distributions and examine the effect of city, geographical region, store chain, and socio-economic status of the targeted clientele on type, cutting style, fat measures and quality of beef cuts of US origin. Top round, bottom round and knuckle were the most common cut types. Milanesa-type slice and "bistec" (steak for grilling) were the predominant cutting styles. Over 95% of the retail cuts were trimmed to 3.2mm or less of external fat. Most cuts were USDA Select (74.5%) and USDA Choice (24.5%). External fat thickness and marbling score differed among cities and store chains (P<0.01).

The active (ADHa) and inactive (ADHi) forms of the PQQ-alcohol dehydrogenase from Gluconacetobacter diazotrophicus differ in their respective oligomeric structures and redox state of their corresponding prosthetic groups.

The membrane-bound alcohol dehydrogenase of Gluconacetobacter diazotrophicus contains one pyrroloquinoline quinone moiety (PQQ), one [2Fe-2S] cluster, and four c-type cytochromes. Here, we describe a novel and inactive enzyme. ADHi, similarly to ADHa, is a heterodimer of 72- and 44-kDa subunits and contains the expected prosthetic groups. However, ADHa showed a threefold molecular mass as compared to ADHi. Noteworthy, the PQQ, the [2Fe-2S] and most of the cytochromes in purified ADHi is in the oxidized form, contrasting with ADHa where the PQQ-semiquinone is detected and the [2Fe-2S] cluster as well as the cytochromes c remained fully reduced after purification. Reduction kinetics of the ferricyanide-oxidized enzymes showed that while ADHa was brought back by ethanol to its full reduction state, in ADHi, only one-quarter of the total heme c was reduced. The dithionite-reduced ADHi was largely oxidized by ubiquinone-2, thus indicating that intramolecular electron transfer is not impaired in ADHi. The acidic pH of the medium might be deleterious for the membrane-bound ADH by causing conformational changes leading to changes in the relative orientation of heme groups and shift of corresponding redox potential to higher values. This would hamper electron transfer resulting in the low activity observed in ADHi.

In vitro effect of malathion and diazinon on oocytes fertilization and embryo development in porcine.

Diazinon and malathion are commonly used organophosphate insecticides in agriculture, industry, and in veterinary medicine as an ectoparasiticide. The importance to carry out in vitro reproductive toxicology assays lies on the need of knowing the alterations these insecticides may cause at cellular level, since they are endocrine disruptors that interfere with reproductive functions. The aim of this study was to evaluate in vitro oocyte viability, fertilization, and embryo development with different concentrations of diazinon and malathion. For in vitro fertilization (IVF), porcine oocytes and sperm were co-incubated for 7 h with increasing concentrations (50, 100, and 500 microM) of diazinon and malathion. For embryo development, fertilized oocytes were cultured in medium containing the same insecticide concentrations during 96 h for embryo development and 144 h for morulae formation. Diazinon did not affect oocyte viability and embryo divisions but decreased IVF (fertilization inhibition(50) = 502 microM) and morulae formation (morulae inhibition(50) = 344 microM). Malathion affected all the studied parameters: lethal concentration(50) = 1 mM, fertilization inhibition(50) = 443 microM, development inhibition(50) = 375 microM, and morulae inhibition(50) = 216 microM. The results of this study indicate that diazinon and malathion used in commercial formulation could be toxic, producing impairment in in vitro fertilization and embryo development. This is an approach for further investigations to find out cell damage mechanisms produced by these widely used insecticides.

The PQQ-alcohol dehydrogenase of Gluconacetobacter diazotrophicus.

The oxidation of ethanol to acetic acid is the most characteristic process in acetic acid bacteria. Gluconacetobacter diazotrophicus is rather unique among the acetic acid bacteria as it carries out nitrogen fixation and is a true endophyte, originally isolated from sugar cane. Aside its peculiar life style, Ga. diazotrophicus, possesses a constitutive membrane-bound oxidase system for ethanol. The Alcohol dehydrogenase complex (ADH) of Ga. diazotrophicus was purified to homogeneity from the membrane fraction. It-exhibited two subunits with molecular masses of 71.4 kDa and 43.5 kDa. A positive peroxidase reaction confirmed the presence of cytochrome c in both subunits. Pyrroloquinoline quinone (PQQ) of ADH was identified by UV-visible light and fluorescence spectroscopy. The enzyme was purified in its full reduced state; potassium ferricyanide induced its oxidation. Ethanol or acetaldehyde restored the full reduced state. The enzyme showed an isoelectric point (pI) of 6.1 and its optimal pH was 6.0. Both ethanol and acetaldehyde were oxidized at almost the same rate, thus suggesting that the ADH complex of Ga. diazotrophicus could be kinetically competent to catalyze, at least in vitro, the double oxidation of ethanol to acetic acid.