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The discovery that the lung harbors a diverse microbiome, as revealed by next-generation sequencing, has significantly altered our understanding of respiratory health and disease. Despite the association between the lung microbiota and disease, the nature of their relationship remains poorly understood, and culture isolation of these microorganisms could help to determine their role in lung physiology. Current procedures for processing samples from the lower respiratory tract have been shown to affect the viability of microorganisms, so it is crucial to develop new methods to improve their survival. This study aimed to improve the isolation and characterization of lung microorganisms using a bead-beating homogenization method in a mouse model. Microsphere diameter and bead-beating time affected the survival of the microorganisms (E. coli, S. aureus and C. albicans). Using 2.3 mm diameter microspheres for 60 s of bead-beating promoted the survival of both bacteria and yeast strains. After intratracheal instillation of these microorganisms in mice, approximately 70% of the cells were recovered after the tissue homogenization. To assess the efficiency of the proposed method, the diversity of bacteria was compared between the homogenate and lung tissue samples. Ninety-one genera were detected in the lung tissue, and 63 in the homogenate. Bacterial genera detected in the homogenate represented 84% of the total abundance of the microbiota identified in the lung tissue. Taken together, these results demonstrate that the tissue homogenization process developed in this study recovered the majority of the microorganisms present in the lung. This study presents a bead-beating homogenization method for effective cultivation of lung tissue microorganisms, which may help to improve the understanding of host-microbe interactions in the lung., (© 2024. The Author(s).)

Background: The prevalence of obesity has increased dramatically in children worldwide. Obesity has been recognized as a risk factor for more serious viral respiratory infections, mainly in adults., Objective: To study the relationship between overnutrition (obesity and overweight) and clinical severity in children hospitalized with acute respiratory infections of viral origin., Methods: One hundred and forty-three clinical records of children between 2 and 18 years old hospitalized for acute respiratory infection at Clínica Dávila (2014-2018) were analyzed, recording the respiratory viruses detected at the time of hospitalization, weight, and height. Nutritional status was estimated using Z score or body mass index, according to age., Results: Eighty-tree3 children (58%) were positive for more than one respiratory virus. The main virus detected in monoinfection was adenovirus (9.8%), followed by respiratory syncytial virus (7.7%) and parainfluenza virus (7.7%). There were no deaths. Patients with obesity presented more days of hospitalization (P = .04), oxygen therapy (P = .03) and mechanical ventilation (P < .001), as well as a higher probability of requiring mechanical ventilation (P = .001) and of ICU admission (P = .003) compared with children with normal weight. Patients with overweight presented more days of mechanical ventilation (P < .001) than patients with normal weight. No significant differences were found between the presence of viral coinfection and nutritional status., Conclusion: Overnutrition is associated with greater severity of viral respiratory infection in hospitalized children., (Copyright © 2023 SEEN and SED. Published by Elsevier España, S.L.U. All rights reserved.)

The COVID-19 pandemic will likely take years to control globally, and constant epidemic surveillance will be required to limit the spread of SARS-CoV-2, especially considering the emergence of new variants that could hamper the effect of vaccination efforts. We developed a simple and robust - Phone Screen Testing ( PoST ) - method to detect SARS-CoV-2-positive individuals by RT-PCR testing of smartphone screen swab samples. We show that 81.3-100% of individuals with high-viral-load SARS-CoV-2 nasopharyngeal-positive samples also test positive for PoST , suggesting this method is effective in identifying COVID-19 contagious individuals. Furthermore, we successfully identified polymorphisms associated with SARS-CoV-2 Alpha, Beta, and Gamma variants, in SARS-CoV-2-positive PoST samples. Overall, we report that PoST is a new non-invasive, cost-effective, and easy-to-implement smartphone-based smart alternative for SARS-CoV-2 testing, which could help to contain COVID-19 outbreaks and identification of variants of concern in the years to come., Competing Interests: RY, AB, CA co-founder of Diagnosis Biotech, CS, AT, ML, EB, CT, AS, FS, NP, LC, ML, NV, AS, AS, FR, GD, LQ No competing interests declared, (© 2021, Young et al.)

Background: Although the nasopharyngeal swab (NPS) is the reference sampling method for the detection of SARS-Cov-2, it is not always possible to collect NPS in some patients. Saliva represents an interesting sampling method because it is less invasive and more convenient in patients with nasal or pharyngeal lesions. Objective: To compare the RT-qPCR test performances of saliva samples with nasal mid-turbinate swab (NMTS) and NPS samples in a cohort of ambulatory patients suspected of having COVID-19. Study Design: For each of the 112 enrolled patients, NPS, NMTS, and saliva samples were collected and tested for SARS-Cov-2 detection using three different target genes (RdRP, N and E genes) by RT-qPCR. Results: Among the positive samples (56/112), saliva samples showed a lower percentage of SARS-Cov-2 detection compared to NPS samples, (85.7 vs . 96.4%), while still a lower percentage was observed for NMTS samples (78.6%). In average, saliva samples showed higher Ct values for all tested target genes, compared to those from NPS and NMTS samples. Conclusions: By using the Allplex TM 2019-nCoV Assay Kit, saliva samples showed lower sensitivity for SARS CoV-2 compared to NPS samples; however, the not detected cases had lower viral burden in NPS samples (CT values >33) representing an interesting alternative sampling method in patients in which it is not possible to take a NPS sample., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tapia, Marcia, Ivone, Nadia, Lesly, Camila, Valentina, Paula and Fabien.)

In the present work, the chemical composition of the Chilean freshwater crab Aegla cholchol exoskeleton was studied for the first time. α-Chitin was isolated from three main body parts (pincers, legs, carapace), and its content ranged from 9.0-10.4% (w/w). Moreover, chitosan was extracted by alkaline treatment at different temperature and time regimes. Aegla cholchol exoskeleton, chitin and chitosans were characterized by FTIR, TGA, and SEM. Chemical structure of chitin and chitosan was confirmed by NMR spectroscopy. Chitosan molecular weight determinations were carried out by GPC. The obtained chitosan samples had a degree of N-acetylation (DA) between 4 and 15% and molecular weight (Mw) in the range of 65-201 kDa. The antifungal activity of the chitosan samples and the chitooligomer were tested toward twenty isolated clinical strains of Candida yeast. Chitosan with lower DA (4%) and higher molecular weight showed the lowest minimum inhibitory concentration (MIC) values, which was evidenced by the highest antifungal activity toward Candida albicans, Candida tropicalis, Candida glabrata, and Candida parapsilosis. Results suggest that Aegla cholchol is an excellent natural source for production of bioactive materials with potential applications in the health system, to prevent infections associated with Candida strains., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Candida albicans is commensal yeast that colonizes skin and mucosa; however, it can become an opportunist pathogen by changing from blastoconidia (commensal form) into hypha (pathogenic form). Each form activates a different cytokines response in epithelial cells. Little is known about the commensal role of C. albicans in the innate immunity. This work studied whether stimulation with C. albicans blastoconidia induces protection in keratinocytes and/or in a reconstituted human epithelium (RHE) infected with C. albicans. For this, inactivated C. albicans blastoconidia was used to stimulate keratinocytes and RHE prior to infection with C. albicans. Blastoconidia induced different cytokine expression profiles; in the case of RHE it decreased interleukin (IL)-1β and IL-10 and increased IL-8, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ). A significant increase in the expression of human β-defensins (HBD) 2 and HBD3 was observed in blastoconidia stimulated keratinocytes and RHE, associated with impaired growth and viability of C. albicans. Additionally, blastoconidia stimulation decreased the expression of virulence factors in C. albicans that are associated with filamentation (EFG1, CPH1 and NRG1), adhesion (ALS5), and invasion (SAP2). Blastoconidia stimulated RHE was significantly less damaged by C. albicans invasion. These results show that the commensal form of C. albicans would exert a protective effect against self-infection., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Objectives: We investigated the colonisation by Candida spp in patients using orthodontic fixed appliances by characterising the isolated Candida strains and by evaluating the host oral mucosa response through the measure of human β-defensins 3 (HBD-3) expression and Interleukin-1ß/IL-10., Methods: Ninety patients were enrolled after signing an informed consent. Prevalence, susceptibility to fluconazole, genotyping and oral fungal burden of Candida sp. isolated were determined. Host responses were evaluated by measuring HBD-3 expression as well as IL-1ß and IL-10 in saliva., Results: The colonisation rate reached 6.7% (6/90), and 5 patients were colonised with C. albicans strains and one with one with C. tropicalis. The fluconazole MIC90/susceptibility of C. albicans strains ranged 1/0.25-1 μg/mL. However, isolated strains did not present different genotype (SAB>0.9), C. albicans colonisation seems to be influenced by the duration of treatment and by level expression of HBD3 that were higher in colonised patients (not statistically different). A negative correlation between the fungal burden and IL-1ß levels was found in colonised patients but not for IL-10., Conclusions: Our study revealed that patients with orthodontic fixed appliances were mainly colonised by C. albicans, which was related to a decrease in HBD-3 expression and IL-1ß levels., (© 2018 Blackwell Verlag GmbH.)

Epithelial cells represent one of the most important physical barriers to many bacterial pathogens. In the case of Neisseria gonorrhoeae, the epithelial cell response is critical because they are the main target of the tissue damage triggered by the pathogen, particularly when the organism reaches the Fallopian tube (FT). Although the irreversible damage triggered by N. gonorrhoeae in the FT has been previously reported (ectopic pregnancy, pelvic inflammatory disease and infertility), the mechanisms of gonococcal-induced tissue damage are not fully understood. In addition, the lack of animal models that efficiently mimic the human disease and the complexity of gonococcus-host interactions make studying gonococcal pathogenesis particularly difficult. The use of human immortalized cells is also limited, since a variety of commercial FT cell lines is not yet available. Finally, the phase and antigenic variation of many gonococcal surface molecules involved in attachment and invasion of epithelial tissues leads to a failure to reproduce results using different human cells lines used in previous studies. The FT organ in culture (FTOC) and primary human fallopian tube epithelial cell (FTEC) represent the closest ex vivo cell models to explore the biology of Neisseria gonorrhoeae during infection of the FT, since it is a natural host target of the gonococcus. In this chapter, we describe protocols to process human FT samples to obtain FTOC and FTEC and assess their response to infection with Neisseria gonorrhoeae.

Melanin is a pigment found in all biological kingdoms, and plays a key role in protection against ultraviolet radiation, oxidizing agents, and ionizing radiation damage. Melanin exerts an antimicrobial activity against bacteria, fungi, and parasites. We demonstrated an antifungal activity of synthetic and human melanin against Candida sp. The members of the Cryptococcus neoformans and C. gattii species complexes are capsulated yeasts, which cause cryptococcosis. For both species melanin is an important virulence factor. To evaluate if cryptococcal and human melanins have antifungal activity against Cryptococcus species they both were assayed for their antifungal properties and physico-chemical characters. Melanin extracts from human hair and different strains of C. neoformans ( n = 4) and C. gattii ( n = 4) were investigated. The following minimum inhibitory concentrations were found for different melanins against C. neoformans and C. gattii were (average/range): 13.7/(7.8-15.6) and 19.5/(15.6-31.2) μg/mL, respectively, for human melanin; 273.4/(125->500) and 367.2/(125.5->500) μg/mL for C. neoformans melanin and 125/(62.5-250) and 156.2/(62-250) μg/mL for C. gattii melanin. Using Scanning Electron Microscopy we observed that human melanin showed a compact conformation and cryptococcal melanins exposed an amorphous conformation. Infrared spectroscopy (FTIR) showed some differences in the signals related to C-C bonds of the aromatic ring of the melanin monomers. High Performance Liquid Chromatography established differences in the chromatograms of fungal melanins extracts in comparison with human and synthetic melanin, particularly in the retention time of the main compound of fungal melanin extracts and also in the presence of minor unknown compounds. On the other hand, MALDI-TOF-MS analysis showed slight differences in the spectra, specifically the presence of a minor intensity ion in synthetic and human melanin, as well as in some fungal melanin extracts. We conclude that human melanin is more active than the two fungal melanins against Cryptococcus. Although some physico-chemical differences were found, they do not explain the differences in the antifungal activity against Cryptococcus of human and cryptococcal melanins. More detailed studies on the structure should be considered to associate structure and antifungal activity.

Objective: To study Candida albicans genotypes using RAPD and their susceptibility to fluconazole in healthy pregnant women and in vulvovaginal candidiasis (VVC) patients after topical treatment with clotrimazole., Methods: Vaginal swabs were collected at t = 0 and t = 1 (1 month later) in pregnant women (control group, n = 33), and before (t = 0), at 1 month (t = 1) and at 2 months (t = 2) after clotrimazole treatment in pregnant women with VVC., Results: Candida albicans was isolated in 30% of healthy pregnant women and 80% of patients with VVC. A high genetic heterogeneity was observed in C. albicans genotypes between individuals. In patients with VVC, topical antifungal treatment with clotrimazole was clinically effective, but only in a 62% C. albicans was eradicated. In patients in which C. albicans was not eradicated, this microorganism persisted for 1 or 2 months after the antifungal treatment. The persistent colonies were not associated with a specific genotype, but they were associated with higher MICs in comparison with colonies isolated from the control group., Conclusions: Therapy with topical clotrimazole, despite a good clinical outcome, could not eradicate completely C. albicans allowing the persistence of genotypes, with higher MICs to fluconazole. More studies with higher number of patients are needed to validate this preliminary finding.

Epithelial shedding and scarring of fallopian tube mucosa are the main consequences of sexually transmitted Neisseria gonorrhoeae infection and probably involve an imbalance of host extracellular matrix components and their regulators such as matrix metalloproteinases (MMPs). In the current study, primary human fallopian tube epithelial cells were infected with N. gonorrhoeae, and MMP patterns were examined. Gonococcal infection induced a significant increase in secreted MMP-9 and an accumulation of cytoplasmic MMP-2 over time, but no significant MMP-3 or MMP-8 production was observed. Thus, MMP-9 in particular could play a role in tubal scarring in response to gonococcal infection., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a 49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE-6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human β-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)