Inhaler Steroid Use Changes Oral and Airway Bacterial and Fungal Microbiome Profile in Asthma Patients.

INTRODUCTION: The full spectrum of bacterial and fungal species in adult asthma and the effect of inhaled corticosteroid use is not well described. The aim was to collect mouthwash and induced sputum samples from newly diagnosed asthma patients in the pretreatment period and in chronic asthma patients while undergoing regular maintenance inhaled corticosteroid therapy, in order to demonstrate the bacterial and fungal microbiome profile. METHODS: The study included 28 asthmatic patients on inhaler steroid therapy, 25 steroid-naive asthmatics, and 24 healthy controls. Genomic DNA was isolated from induced sputum and mouthwash samples. Analyses were performed using bacterial primers selected from the 16S rRNA region for the bacterial genome and "panfungal" primers selected from the 5.8S rRNA region for the fungal genome. RESULTS: Dominant genera in mouthwash samples of steroid-naive asthmatics were Neisseria, Haemophilus, and Rothia. The oral microbiota of asthmatic patients on inhaler steroid treatment included Neisseria, Rothia, and Veillonella species. Abundant genera in induced sputum samples of steroid-naive asthma patients were Actinomyces, Granulicatella, Fusobacterium, Peptostreptococcus, and Atopobium. Sputum microbiota of asthma patients taking inhaler steroids were dominated by Prevotella and Porphyromonas. Mucor plumbeus and Malassezia restricta species were abundant in the airways of steroid-naive asthma patients. Choanephora infundibulifera and Malassezia restricta became dominant in asthma patients taking inhaled steroids. CONCLUSION: The oral and airway microbiota consist of different bacterial and fungal communities in healthy and asthmatic patients. Inhaler steroid use may influence the composition of the oral and airway microbiota.

COVID-19-associated pulmonary aspergillosis (CAPA): identification of Aspergillus species and determination of antifungal susceptibility profiles.

Among the co-infectious agents in COVID-19 patients, Aspergillus species cause invasive pulmonary aspergillosis (IPA). IPA is difficult to diagnose and is associated with high morbidity and mortality. This study is aimed at identifying Aspergillus spp. from sputum and tracheal aspirate (TA) samples of COVID-19 patients and at determining their antifungal susceptibility profiles. A total of 50 patients with COVID-19 hospitalized in their intensive care units (ICU) were included in the study. Identification of Aspergillus isolates was performed by phenotypic and molecular methods. ECMM/ISHAM consensus criteria were used for IPA case definitions. The antifungal susceptibility profiles of isolates were determined by the microdilution method. Aspergillus spp. was detected in 35 (70%) of the clinical samples. Among the Aspergillus spp., 20 (57.1%) A. fumigatus, six (17.1%) A. flavus, four (11.4%) A. niger, three (8.6%) A. terreus, and two (5.7%) A. welwitschiae were identified. In general, Aspergillus isolates were susceptible to the tested antifungal agents. In the study, nine patients were diagnosed with possible IPA, 11 patients were diagnosed with probable IPA, and 15 patients were diagnosed with Aspergillus colonization according to the used algorithms. Serum galactomannan antigen positivity was found in 11 of the patients diagnosed with IPA. Our results provide data on the incidence of IPA, identification of Aspergillus spp., and its susceptibility profiles in critically ill COVID-19 patients. Prospective studies are needed for a faster diagnosis or antifungal prophylaxis to manage the poor prognosis of IPA and reduce the risk of mortality.

A Novel Intronic Mutation Reduces HAX1 Level and is Associated With Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a rare disease. Autosomal recessive forms of SCN are more frequent in countries where consanguineous marriages are common. In this report, we describe a 54-day-old female with neutropenia who presented with ecthyma gangrenosum. Clinical exome sequencing was used to identify the mutation. HAX1 messenger RNA and isoforms were examined by real-time quantitative and conventional polymerase chain reaction. Bone marrow aspiration was stained by hematoxylin and eosin. Granulocytes were tested for apoptosis upon H2O2 exposure. T-cell proliferation was tested by flow cytometry. Clinical exome sequencing revealed a novel homozygous acceptor splice site mutation in intron 3 of HAX1 (c.505-1G>C), which reduced both isoforms A and B of HAX1 messenger RNA. The Western blot studies showed a complete absence of HAX1 protein. The purified neutrophils from the patient showed increased apoptosis upon H2O2 exposure, whereas T-cell proliferative responses to various stimuli were intact. The patient was treated with combined antibiotics, filgrastim, and placed on antibiotics prophylaxis. To the best of our knowledge, our data provide the first experimental evidence for HAX1 deficiency because of a splice site mutation. Although 3 other splice site variants have been deposited in databases, functional studies were missing. This novel variant of HAX1 may explain the SCN and secondary infections in our patients.

Quantitation of antibodies against SARS-CoV-2 spike protein after two doses of CoronaVac in healthcare workers.

Quantitation of antibodies to the spike protein of severe acute respiratory syndrome coronavirus 2  (SARS-CoV-2) was performed for the detection of adaptive immune response in healthcare workers (HCWs) vaccinated with CorovaVac. We prospectively recruited HCWs from a university hospital in Turkey. Serum samples from 1072 HCWs were obtained following 28 days of the first, and 21 days of the second dose. Detection and quantitation of SARS-CoV-2 antispike antibodies were performed by the chemiluminescent microparticle immunoassay (SARS-CoV-2 IgG II Quant; Abbott). Results greater than or equal to the cutoff value 50.0 AU/ml were reported as positive. After the first dose, antispike antibodies were detected in 834 of 1072 (77.8%) HCWs. Seropositivity was higher among females (84.6%) than males (70.6%) (p < 0.001) and was found to be highest in both women and men between the ages of 18-34. After the second dose, antibodies were detected in 1008 of 1012 (99.6%) HCWs. Antibody titers were significantly higher in those who had coronavirus disease-2019 before vaccination than those who did not (p < 0.001). Antibody positivity and median antibody titers were significantly less in HCWs with chronic diseases compared to those without (p < 0.05 and p < 0.001, respectively). In conclusion, our findings indicated that a relatively high frequency (99.6%) of humoral immunity was produced in HCWs aged 18-59 after two doses of CoronaVac. Quantitation of antibodies may help facilitate longitudinal monitoring of the antibody response, which will be especially useful in deciding the dose of the vaccine in vulnerable groups such as those over 60 years of age and those with chronic diseases.

Design, synthesis and biological evaluation of new benzoxazolone/benzothiazolone derivatives as multi-target agents against Alzheimer's disease.

In this study, four series of compounds with benzoxazolone and benzothiazolone cores were designed, synthesized and evaluated as multifunctional agents against Alzheimer's disease (AD). Additionally, in order to shed light on the effect of the carbonyl groups of benzoxazolone/benzothiazolone, benzoxazole/benzothiazole-containing analogues were also synthesized and evaluated. Inhibition potency of all final compounds towards cholinesterase enzymes and their antioxidant activity were tested. Subsequently, the anti-inflammatory activity, cytotoxicity, apoptosis, and Aß aggregation inhibition tests were also performed for selected compounds. The results indicated that compounds 11c, a pentanamide derivative with benzothiazolone core, and 14b, a keton derivative with benzothiazolone core, were considered as promising multi-functional agents for further investigation against AD. The reversibility, kinetic and molecular docking studies were also performed for the compounds with the highest AChE 14b (eeAChE IC50 = 0.34 µM, huAChE IC50 = 0.46 µM) and BChE 11c (eqBChE IC50 = 2.98 µM, huBChE IC50 = 2.56 µM) inhibitory activities.

Succinivibrionaceae is dominant family in fecal microbiota of Behçet's Syndrome patients with uveitis.

Behçet's Syndrome (BS) is a multisystem vasculitis with various clinical manifestations. Pathogenesis is unclear, but studies have shown genetic factors, innate immunity and autoinflammation to have an important role in the disease course. Diversity in the microbial community of gut microbiota may significantly contribute to the activation of the innate immune system. The clinical features of BS present themselves in clusters and each cluster may be a consequence of different disease mechanisms. For this reason we aimed to investigate the gut microbiota of BS patients with uveitis. In addition to healthy controls, we have aimed to compare the gut microbiota of BS with that of Familial Mediterranean Fever (FMF) and Crohn's Disease (CD) as both diseases have innate and autoinflammatory features in their pathogenesis. Seven patients with BS, 12 patients with FMF, 9 patients with CD and 16 healthy controls (HC) were included in the study. Total genomic DNAs were isolated from fecal samples of the patients. Partial 16S rRNA gene was sequenced using the PGM Ion Torrent (Thermo Fisher Scientific, Waltham, MA, USA) for microbiota analysis. Statistical analysis showed that significant differences were detected on the microbial community of four groups. Succinivibrionaceae is dominant and the signature family, whereas Bacteroides was absent in BS patients.

Comparison of corneal collagen cross-linking (PACK-CXL) and voriconazole treatments in experimental fungal keratitis.

PURPOSE: To compare the antifungal efficacy of corneal collagen cross-linking with photoactivated riboflavin (PACK-CXL) and voriconazole in experimental Fusarium solani and Candida albicans keratitis models. METHODS: Sixty-four corneas of 32 New Zealand rabbits were included and divided into two main groups. Intrastromal injection of Fusarium and Candida suspensions was performed, and it was observed that keratitis was formed on the third day. Both groups were randomly separated into the following four groups: control, PACK-CXL, voriconazole and PACK-CXL combined with voriconazole. PACK-CXL was applied using 0.25% riboflavin in an accelerated Dresden protocol (total ultraviolet A dose 5.4 J/cm²). Voriconazole was applied topically as 7x1/day with a dose of 1% (10 mg/ml). Corneal buttons were excised on the tenth day, and microbiological and pathological examinations were performed. RESULTS: The PACK-CXL and PACK-CXL combined with voriconazole groups each had 100 colony-forming unit (CFU/ml) of reproduced micro-organisms compared with 500 CFU/ml in the voriconazole group and 1500 CFU/ml in the control group (p < 0.001) in the Fusarium keratitis model. The PACK-CXL combined with voriconazole group had 100 CFU/ml, the PACK-CXL group had 150 CFU/ml, and the voriconazole group had 200 CFU/ml of reproduced micro-organisms compared with 4000 CFU/ml in the control group (p < 0.002) in the Candida keratitis model. (p < 0.001). Fewer hyphae and non-specific stromal changes were observed in the pathological cross sections examined in subgroups that used CXL. CONCLUSION: There was less fungus reproduction and a lower keratitis score for Fusarium solani and Candida albicans in the treatment groups compared to the control groups, especially in groups that used PACK-CXL. These results suggest that it is useful to combine PACK-CXL treatment with medical treatment in the fungal keratitis algorithm at the early stage of the disease.

Design, Synthesis and Investigation of New Diphenyl Substituted Pyridazinone Derivatives as Both Cholinesterase and Aß-Aggregation Inhibitors.

BACKGROUND: With respect to the increase in the average life expectancy, Alzheimer Disease (AD), the most common form of age-related dementia, has become a major threat to the population over the age of 65 during the past several decades. The majority of AD treatments are focused on cholinergic and amyloid hypotheses. OBJECTIVE: In this study, three series of diphenyl-2-(2-(4-substitutedpiperazin-1-yl)ethyl)pyridazin- 3(2H)-one derivatives were designed, synthesized and investigated for their ability to inhibit both cholinesterase enzymes and amyloid-ß aggregation. METHOD: The inhibitory activities of the synthesized compounds on AChE (from electric eel) and BChE (from equine serum) were determined by the modified Ellman's method. The reported thioflavin T-based fluorometric assay was performed to investigate the effect of the selected compounds on the aggregation of Aß1-42. The cytotoxic effect of the compounds (4g, 11g and 18g) was monitored in 3T3 cell lines to gain insight into therapeutic potential of the compounds by using MTT assay. The crystal structures of the AChE (1EVE) and BChE (1P0I) enzymes were retrieved from the RCSB Protein Data Bank and Molecular Operating Environment (MOE) software was used for molecular docking of the ligands. RESULTS: Among the tested compounds, 5,6-diphenyl derivative 18g was identified as the most potent and selective AChE inhibitor (IC50 = 1.75 µM, Selectivity Index for AChE > 22.857). 4,6- Diphenyl derivative 11g showed the highest and the most selectivity for BChE (IC50= 4.97 µM, SI for AChE < 0.124). Interestingly, 4,5-diphenyl derivative 4g presented dual cholinesterase inhibition (AChE IC50= 5.11 µM; BChE IC50= 14.16 µM, SI for AChE = 2.771). CONCLUSION: Based on biological activity results and low toxicity of the compounds, it can be said that diphenyl substituted pyridazinone core is a valuable scaffold. Especially, dual inhibitory potencies of 4,5-diphenylpyridazin-3(2H)-one core for the cholinesterase enzymes and Aß- aggregation makes this core a promising disease-modifying agent.

Impact of Iontophoresis and PACK-CXL Corneal Concentrations of Antifungals in an In Vivo Model.

PURPOSE: To investigate voriconazole (VRZ) penetration and fungal load in the cornea after applying VRZ therapy with various treatment combinations in a fungal keratitis model. METHODS: Fifty-four eyes of 27 young albino rabbits were provided for this experimental study. Twelve corneas were inoculated with Candida albicans, 12 corneas were inoculated with Fusarium solani, and 6 eyes were selected as controls. Infected corneas received various treatment combinations including VRZ 1% drop therapy alone, VRZ 1% plus amphotericin B 1% drop combination therapy, iontophoretic VRZ therapy, and VRZ 1% drop therapy after corneal cross-linking. Fungal load was measured by log reduction, and VRZ levels were quantified by liquid chromatography-tandem mass spectrometry. RESULTS: Iontophoresis-assisted VRZ application showed the highest antifungal activity against F. solani keratitis (4-log reduction) and C. albicans keratitis (5-log reduction) compared with other treatment applications. VRZ levels were also found to be the highest in corneas that received iontophoretic VRZ treatment (3.6313 ± 0.0990 ppb for F.solani keratitis and 1.7001 ± 0.0065 ppb for C. albicans keratitis) compared with other treatment applications. CONCLUSIONS: Iontophoresis seems to provide the highest VRZ concentration and highest antifungal activity in the cornea compared with other treatment applications for C. albicans and F. solani keratitis.

The in vitro effect of antimicrobial photodynamic therapy on Candida and Staphylococcus biofilms

Background/aim: This study was designed to evaluate the effect of antimicrobial photodynamic treatment (APDT) in a biofilm model using combinations of various dyes (rose bengal, riboflavin, and methylene blue) as photosensitizers and light sources (LED and UVA) against staphylococcal and candidal biofilms. Materials and methods: Sterile microtiter plates were used for the development and quantification of the biofilms. APDT was carried out using combinations of the light sources and dyes. The percentage of the growth inhibition was then calculated using a spectrophotometer. The broth media in the wells were aspirated, wells were stained with crystal violet, and optical density values were measured spectrophotometrically. SEM analysis of the impact of APDT on bacterial and fungal biofilms was also performed. Results: The experiments showed that the most efficacious combination was red LED + methylene blue against both staphylococcal and candidal biofilms. A marked inhibition (45.4%) was detected on both C. albicans and C. parapsilosis biofilms. Red LED + methylene blue was also effective on S. aureus and S. epidermidis biofilms. SEM images suggested that the number of adherent cells and biofilm mass were markedly reduced after APDT treatment. Conclusion: Although the results of this study indicated the in vitro efficacy of APDT, it might also be a promising technique for the control of biofilm growth within intravenous catheters.

Corneal Cross-Linking Has No Effect on Matrix Metalloproteinase 9 and 13 Levels During Fungal Keratitis on the Early Stage.

The aim of our study was to investigate matrix metalloproteinases, MMP-9 and MMP-13 levels, in the rabbit model of Fusarium and Candida keratitis treated by corneal cross-linking (PACK-CXL). Rabbit corneas were inoculated with fungal inoculum for keratitis. Each group divided into four subgroups, including un-treated group, PACK-CXL group, voriconazole group and PACK-CXL plus voriconazole group. PACK-CXL was applied with 0.25% riboflavin in accelerated Dresden protocol, and 0.1% voriconazole drops were administered. All corneal buttons excised at tenth day after ophthalmological examination. Fungal cell counts and Scheiber scores were determined in all groups. Corneal tissue MMP mRNA levels were evaluated quantitative reverse transcriptase PCR. The difference in MMP-9 and MMP-13 levels at all groups was not statistically significant (p > 0.05). PACK-CXL with 0.25% riboflavin either alone or combined with antifungal drops was unable to provide decline in inflammatory findings in both macroscopic and microscopic levels similar to medical antifungal treatment.

[Using Galleria mellonella as an in vivo model to study the virulence of some bacterial and fungal agents]. / Bazi bakteri ve mantarlarin virülansinin arastirilmasinda Galleria mellonella'nin in vivo model olarak kullanilmasi.

Non-vertebrate hosts, such as Galleria mellonella, namely wax moth, have been used to study microbial virulence and host defense. This organism has advantages as it is economical, ethically expedient and easy to handle. Here we describe an experimental in vivo study using the larvae of Galleria mellonella infected with some bacterial and fungal pathogens. In this study, extended-spectrum beta-lactamase (ESBL) producing and non-producing Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, colistin resistant and susceptible Acinetobacter baumanii clinical strains; Candida albicans (ATCC 10231), Scedosporium aurantiacum (CBS 136047) and Pseudallescheria boydii (CBS 117410) reference strains, and Aspergillus terreus and Fusarium oxysporum clinical strains were used as pathogens. The larvae of G.mellonella were challenged with these bacterial and fungal strains, and the mortality rates were calculated using Kaplan-Meier plots. Mortality rates at 16th hour were found as 83% for the larvae infected with both ESBL positive and negative E.coli, ESBL negative K.pneumoniae and ESBL positive P.aeruginosa; 91% for ESBL positive K.pneumoniae; 75% for ESBL negative P.aeruginosa; 66% for both colistin resistant and susceptible A.baumanii strains. All larvae infected with bacteria died within the first 24 hour. Larvae infected with bacteria showed significantly higher mortality rates than those infected with fungi. Mortality rates at 16th hour were found as 0% for C.albicans and F.oxysporum, 16% for S.aurantiacum, 8% for P.boydii and A.terreus; at 24th hour that was 25% for C.albicans and P.boydii, 33% for S.aurantiacum, A.terreus and F.oxysporum; at 48th hour that was 33% for C.albicans, 50% for P.boydii and F.oxysporum, 58% for A.terreus, and 66% for S.aurantiacum; in 72 hours that was 58% for C.albicans and F.oxysporum, 66% for P.boydii, 75% for A.terreus and S.aurantiacum, in 96 hours that was 83% for C.albicans, P.boydii and F.oxysporum, 91% for A.terreus and S.aurantiacum. As a result of this study, potential evidences provided that bacteria were more virulent than fungi for G.mellonella larvae model, each fungal species showed different virulence patterns, and bacterial virulence was correlated neither with species nor antibiotic susceptibility.