Biofilm analyses and exoproduct release by clinical and environmental isolates of Burkholderia pseudomallei from Brazil

Objective:To characterize biofilm production by clinical(n=21)and environmental(n=11)isolates of Burkholderia pseudomallei and evaluate the production of proteases,hemolysins and siderophores.Methods:Initially,the 32 strains were evaluated for biofilm production in Müller-Hinton broth-1%glucose(MH-1%glucose)and BHI broth-1%glucose,using the crystal violet staining technique.Subsequently,growing(48 h)and mature(72 h)biofilms were evaluated by confocal microscopy.Finally,the production of proteases,hemolysins and siderophores by planktonic aggregates,growing biofilms and mature biofilms was evaluated.Results:All isolates produced biofilms,but clinical isolates had significantly higher biomass in both MH-1%glucose(P<0.001)and BHI-glucose 1%(P=0.005).The structural analyses by confocal microscopy showed thick biofilms,composed of multiple layers of cells,homogeneously arranged,with mature biofilms of clinical isolates presenting higher biomass(P=0.019)and thickness of the entire area(P=0.029),and lower roughness coefficient(P=0.007)than those of environmental isolates.Protease production by growing biofilms was significantly greater than that of planktonic(P<0.001)and mature biofilms(P<0.001).Hemolysin release by planktonic aggregates was higher than that of biofilms(P<0.001).Regarding siderophores,mature biofilms presented higher production than growing biofilms(P<0.001)and planktonic aggregates(P<0.001).Conclusions:Clinical isolates have higher production of biofilms than their environmental counterparts;protease and siderophores seem important for growth and maintenance of Burkholderia pseu­domallei biofilms.

eHealth,telehealth,and telemedicine in the management of the COVID-19 pandemic and beyond:Lessons learned and future perspectives

In this article,we discuss evidence supporting the effective implementation of e Health,telehealth,and telemedicine during the coronavirus disease 2019 pandemic,with a view towards its permanent future integration in healthcare.We performed a literature search for articles describing the use of telehealth/telemedicine in the pandemic context using five databases.The articles selected describe the use of telemedicine as its advantages in terms of practicality and costeffectiveness.This synthesis of articles is applicable to high-,middle-and lowincome countries.Some of the notable benefits include breaking down geographical and time barriers,reducing waiting lists and crowding in healthcare facilities,and saving on national healthcare expenditure.However,there are a number of difficulties with the widespread implementation of telemedicine services that mainly relate to bureaucratic and regulatory concerns.Moreover,it is also important to make healthcare professionals and providers aware of the limits of this tool to avoid potential cases of negligence.Patients in turn will have to be made aware of and be educated on the use of this new healthcare modality before it is accepted by them.In the current socio-economic climate,it is therefore essential to implement a telehealth model aimed at efficiency and continuity of healthcare,as well as leading to an improvement in the quality of life of patients,whilst optimising existing resources and reducing costs.In that regard,the adoption of e Health,telehealth,and telemedicine services should be considered highly timely,despite current existing limitations.

Relationship between Methylenetetrahydrofolate Reductase (C677T), Factor V Leiden (G1691A), Prothrombin Mutation (G20210A) and Severe Preeclampsia in a Brazilian Population

Objectives: To better understand the etiologic factors that can influence preeclampsia, we inves- tigated hereditary factors for thrombosis, FV Leiden, F II 20210A mutation and the polymorphism C677T of the MTHFR, as singly and as in association, in a group of women from Ceará state-Northeast Brazil with severe preeclampsia. Material and Methods: We conducted a case-control study. 101 cases of severe preeclampsia were recruited from School Maternity Assis Chateaubriand, a reference Maternity of State of Ceará, Brazil, from December 2009 to December 2010. For clinical correlations, women were interrogated about fetal low weight (less than2500 grams) and fetal loss in previous pregnancies. Low weight, fetal loss and necessity of neonatal intensive care unit (neonatal ICU) related to current pregnancy were registered. 245 healthy voluntary women were recruited from the blood bank donors to verify the frequency of FV Leiden, FII and MTHFR as a control group. The study was approved by the Committee on Ethics in Human Research of Maternidade Escola Assis Chateubriand and all individuals gave their informed consent to participate in the study. Standard veinipuncture, with EDTA as anticoagulant, was used to collect blood samples. Genomic DNA was extracted, soon after, using a salting out method. Agarose gel electrophoresis with ethidium bromide staining was performed to ensure the quality of DNA extraction. Results: The frequencies of FV Leiden and FII mutation carrier were 0.99% (1/101) for both factors in the preeclampsia patients and 1.86% (4/214) and 0.93% (2/214), respectively, in control group. All mutates were heterozygous and concomitance of both mutations was not found. The genotype distribution of the MTHFR C677T in the patients and controls frequencies was in Hardy-Weinberg equilibrium (p ≤ 0.05). No statistical difference was observed between cases and controls in MTHFR genotypes or alleles. Conclusions: The FV Leiden, FII G20210A mutation and MTHFR C677T were not risks for preeclampsia development. FV Leiden and FII G20210A mutations had low frequency in the population studied, which may justify the absence of association.

Research advances on the multiple uses of Moringa oleifera: a sustainable alternative for socially neglected population

Moringa oleifera Lam(Moringaceae) is a plant with high nutritional and medicinal value. Native to India, it is now widely distributed throughout tropical and subtropical regions of the world. Its different parts are sources of proteins, vitamins and minerals and present different pharmacological and biotechnological potential. Moreover, M. oleifera seeds are widely used in water and effluent treatment, for their coagulation, flocculation and sedimentation properties, their ability of improving water quality, by reducing organic matter and microbial load, with special applicability in intensive animal production systems, such as aquaculture. In addition, due to its high nutritional value and several medicinal properties, this tree may act as a nutritional and medical alternative for socially neglected populations. In this context, this review gathers information on M. oleifera, emphasizing its chemical constituents, nutritional, pharmacological and antimicrobial properties, applications in the treatment of water effluents, and ecological and social aspects.

Enterobacteria and Vibrio from Macrobrachium amazonicum prawn farming in Fortaleza,Ceará,Brazil

Objective:To investigate the isolation of enterobacteria associated with Macrobrachium amazonicum(M.amazonicum) farming and evaluate the in vitro antimicrobial susceptibility of Vibrio strains.Methods:Strains were isolated from female M.amazonicum prawns and environmental and hatchery water.Biochemical assays were used to identify bacterial genera and those belonging to the genus Vibrio were submitted to further analyses for species identification,through Vitek 2 automated system and scrotyping.Susceptibility test was performed according to Clinical Laboratory Standards Institute.Results:The following genera of enlerobacteria were recovered:Enterobacter(n=11),Citrobacter(n=10),Proteus(n=2),Serratia(n=2),Kluyvera(n=2),Providencia(n=2),Cedecea(n=1),Escherichia(n=1).Edwardsiella(n=1) and BulliauxeUa(n=1).As for Vibrio,three species were identified:Vibrio cholerae non-O1/non-O139(n=4).Vibrio vulnificus(V.vulnificus)(n=1) and Vibrio mimicus(n=1).Vibrio spp.showed minimum inhibitory concentrations values within the susceptibility range established by Clinical Laboratory Standards Institute for almost all antibiotics,except for V.vulnificus,which presented intermediate profile to ampicillin.Conclusions:Enterobacteria do not seem to be the most important pathogens associated with M.amazonicum farming,whereas the recovery of Vibrio spp.from larviculture,with emphasis on Vibrio cholerae and V.vulnificus,deserves special attention due to their role as potentially zoonotic aquaculture-associated pathogens.Furthermore,the intermediate susceptibility of V.vulnificus to ampicillin reflects the importance of monitoring drug use in prawn farming.

P2X7 receptor blockade decreases inflammation,apoptosis,and enteric neuron loss during Clostridioides difficile toxin A-induced ileitis in mice

Clostridioides difficile(C.difficile)is the most common pathogen causing health care-associated infections.C.difficile TcdA and TcdB have been shown to activate enteric neurons;however,what population of these cells is more profoundly influenced and the mechanism underlying these effects remain unknown.AIM To characterize a specific population of TcdA-affected myenteric neurons and investigate the role of the P2X7 receptor in TcdA-induced ileal inflammation,cell death,and the changes in the enteric nervous system in mice.METHODS Swiss mice were used to model TcdA-induced ileitis in ileal loops exposed to TcdA(50μg/Loop)for 4 h.To investigate the role of the P2X7 receptor,Brilliant Blue G(50 mg/kg,i.p.),which is a nonspecific P2X7 receptor antagonist,or A438079(0.7μg/mouse,i.p.),which is a competitive P2X7 receptor antagonist,were injected one hour prior to TcdA challenge.Ileal samples were collected to analyze the expression of the P2X7 receptor(by quantitative real-time polymerase chain reaction and immunohistochemistry),the population of myenteric enteric neurons(immunofluorescence),histological damage,intestinal inflammation,cell death(terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end labeling),neuronal loss,and S100B synthesis(immunohistochemistry).RESULTS TcdA upregulated(P<0.05)the expression of the P2X7 receptor gene in the ileal tissues,increasing the level of this receptor in myenteric neurons compared to that in control mice.Comparison with the control mice indicated that TcdA promoted(P<0.05)the loss of myenteric calretinin+(Calr)and choline acetyltransferase+neurons and increased the number of nitrergic+and Calr+neurons expressing the P2X7 receptor.Blockade of the P2X7 receptor decreased TcdAinduced intestinal damage,cytokine release[interleukin(IL)-1β,IL-6,IL-8,and tumor necrosis factor-α],cell death,enteric neuron loss,and S100B synthesis in the mouse ileum.CONCLUSION Our findings demonstrated that TcdA induced the upregulation of the P2X7 receptor,which promoted enteric neuron loss,S100B synthesis,tissue damage,inflammation,and cell death in the mouse ileum.These findings contribute to the future directions in understanding the mechanism involved in intestinal dysfunction reported in patients after C.difficile infection.

Vibrio spp.from Macrobrachium amazonicum prawn farming are inhibited by Moringa oleifera extracts

Objective:To investigate the in vitro antimicrobial potential of extracts of stem,leaves,flowers,pods and seeds of Moringu okifera(M.okifera) against Vibrio spp.from hatchery water and the prawn Mmrobrachium amazonicum.Methods:The ethanol extracts of stem,leaves,pods and seeds and chloroform extract of flowers of M.oleifera were tested against Vibrio cholerue(V.cholerae) serogroups non-O1/non-O139(n=4).Vibrio vulnificus(n=l) and Vibrio mimicus(n=1).Escherichia coli(E.coli)(ATCC(?) 25922) was used as quality control.Vibrio species were obtained from Macmbrachium amazonicum prawns and from hatchery water from prawn farming.The Minimum Inhibitory Concentration(MIC) was determined by broth microdilution method.Results:The best result was obtained with the ethanol extract of pods,which inhibited three strains of the V.choleme,Vibrio vulnificus,Vibrio minimis and E.coli(MIC range 0.312-5.000 mg/mL).The chloroform extract of flowers was effective against all V.chokrae strains and E.coli(MIC range 0.625-1.250 mg/mL).However,the ethanol extracts of stem and seeds showed low effectiveness in inhibiting the bacterial growth.Conclusions:The extracts of pods,flowers and leaves of M.oleifera have potential for the control of Vibrio spp.Further studies are necessary to isolate the bioaetive compounds responsible for this antimicrobial activity.

Bone regeneration with adipose derived stem cells in a rabbit model

It has been shown that stem cells are able to calcify both in vitro and in vivo once implanted under the skin, if conveniently differentiated. Nowadays, however, a study on their efficiency in osseous regeneration does not exist in scientific literature and this very task is the real aim of the present experimentation. Five different defects of 6 mm in diameter and 2 mm in depth were created in the calvaria of 8 white New Zealand rabbits. Four defects were regenerated using 2 different conveniently modified scaffolds(Bio-Oss Block and Bio-Oss Collagen, Geistlich),with and without the aid of stem cells. After the insertion, the part was covered with a collagen membrane fixed by 5 modified titan pins(Altapin). The defect in the front was left empty on purpose as an internal control to each animal.Two animals were sacrificed respectively after 2, 4, 6, 10 weeks. The samples were evaluated with micro-CT and histological analysis. Micro-CT analysis revealed that the quantity of new bone for samples with Bio-Oss Block and stem cells was higher than for samples with Bio-Oss Block alone. Histological analysis showed that regeneration occurred in an optimal way in every sample treated with scaffolds. The findings indicated that the use of adult stem cells combined with scaffolds accelerated some steps in normal osseous regeneration.

Comparison of p53 and prohibitin expression in the spectrum of hepatitis, cirrhosis, and hepatocellular carcinoma

Aim:To investigate the correlation of p53 and prohibitin (PHB) expression in the spectrum of hepatitis, cirrhosis, and human hepatocellular carcinoma (HCC).Methods:Hepatic biopsies from patients with HCC (n = 60), cirrhosis (CIR,n= 30), hepatitis C virus (HCV,n = 30), and normal livers (NL,n = 20) were examined for immunohistochemical expression of RelA/p65, tumor necrosis factor receptor-1 (TNFR1), TNF-related apoptosis-inducing ligand (TRAIL), p53, and PHB. The samples were also analysed by nuclear factor kappa B (NFκB) Southwestern histochemistry and a transferase-mediated dUTP-biotin nick-end labelling assay.Results: Expression of NFκB and RelA/p65 was detected increasingly from NL to CIR, but had a diminished labelling in the HCC cases (P < 0.05). Expression levels of TNFR1 and TRAIL followed the same pattern (P < 0.05). Apoptosis was increased in HCC, but was progressively reduced from CIR to NL (P < 0.05). P53 and PHB nuclear expressions were ampliifed in cases of HCC, but diminished in NL, HCV, and CIR (P< 0.05).Conclusion: These results suggest that in addition to well-understood sequences of proinlfammatory events such as TNF-induced NFκB activation and NFκB/TRAIL pathway-mediated apoptosis, development of HCC is also inlfuenced by regulation ofp53 andPHB tumour suppressor function. Additional studies are necessary to explain the contradictory mechanisms of the tumour microenvironment observed in the sequence of HCV, CIR, and HCC.