Pore-forming peptide C14R exhibits potent antifungal activity against clinical isolates of Candida albicans and Candida auris.

Introduction: Invasive candidiasis is a global public health problem as it poses a significant threat in hospital-settings. The aim of this study was to evaluate C14R, an analog derived from peptide BP100, as a potential antimicrobial peptide against the prevalent opportunistic yeast Candida albicans and the emergent multidrug-resistant yeast Candida auris. Methods: Antifungal susceptibility testing of C14R against 99 C. albicans and 105 C. auris clinical isolates from Colombia, was determined by broth microdilution. Fluconazole was used as a control antifungal. The synergy between C14R and fluconazole was assessed in resistant isolates. Assays against fungal biofilm and growth curves were also carried out. Morphological alterations of yeast cell surface were evaluated by scanning electron microscopy. A permeability assay verified the pore-forming ability of C14R. Results: C. albicans and C. auris isolates had a geometric mean MIC against C14R of 4.42 µg/ml and 5.34 µg/ml, respectively. Notably, none of the isolates of any species exhibited growth at the highest evaluated peptide concentration (200 µg/ml). Synergistic effects were observed when combining the peptide and fluconazole. C14R affects biofilm and growth of C. albicans and C. auris. Cell membrane disruptions were observed in both species after treatment with the peptide. It was confirmed that C14R form pores in C. albicans' membrane. Discussion: C14R has a potent antifungal activity against a large set of clinical isolates of both C. albicans and C. auris, showing its capacity to disrupt Candida membranes. This antifungal activity remains consistent across isolates regardless of their clinical source. Furthermore, the absence of correlation between MICs to C14R and resistance to fluconazole indicates the peptide's potential effectiveness against fluconazole-resistant strains. Our results suggest the potential of C14R, a pore-forming peptide, as a treatment option for fungal infections, such as invasive candidiasis, including fluconazole and amphotericin B -resistant strains.

The Effects of Nature Exposure Therapies on Stress, Depression, and Anxiety Levels: A Systematic Review.

BACKGROUND: Mental well-being plays a pivotal role within the broader spectrum of health and illness, encompassing factors such as stress, depression, and anxiety. Nature-based therapeutic interventions have emerged as a promising approach to addressing these mental health challenges. This study seeks to assess the impact of these interventions on stress, depression, and anxiety levels. METHODS: We conducted an extensive search for randomized clinical trials that examined stress, anxiety, and depression levels. The selected studies underwent a rigorous risk-of-bias assessment following the guidelines outlined in the Cochrane Handbook for Systematic Reviews. RESULTS: Our review encompassed findings from eight publications. Among them, two studies measuring cortisol levels revealed significant differences between the pre-test and post-test measurements within the intervention groups. In two studies that employed the Stress Response Inventory, a significant decrease in stress levels was observed within the intervention groups in contrast to the control groups. However, no significant differences were noted in studies that utilized the Restorative Outcome Scale. In the assessment of anxiety and depression levels, three studies employed the Positive and Negative Affect Schedule, while four studies utilized The Profile of Mood States scale; none of these studies demonstrated significant differences. CONCLUSIONS: The current body of evidence offers limited support for advocating nature-based therapeutic interventions as a primary approach to reducing stress, depression, and anxiety.

The structure of associations: Method insights from analyzing 28 clinical isolates of Cryptococcus neoformans.

Clinical isolates of a fungal pathogen from a single region or country often exhibit structural clonality or phylogenetic clustering at the sequence or MLST level; such population structure can persist also in larger samples. In efforts to improve causal understanding of pathogenesis at the molecular level, genome-wide association screening methods initially designed for other kingdoms have been applied to fungi. The example of a Colombian dataset of 28 clinical Cryptococcus neoformans VNI isolates indicates where the output from standard pipelines may need to be analyzed in new ways in order to efficiently extract hypotheses for experiments from fungal genotype-phenotype data.

Collections of clinical isolates of a human fungal pathogen can consist of clusters of genetically similar isolates. Such clustering complicates the screening for genetic associations with clinically relevant traits. We propose new methods, illustrating them for the fungus causing cryptococcosis.

A Landscape of the Genomic Structure of Cryptococcus neoformans in Colombian Isolates.

Cryptococcus neoformans species complexes are recognized as environmental fungi responsible for lethal meningoencephalitis in immunocompromised individuals. Despite the vast knowledge about the epidemiology and genetic diversity of this fungus in different regions of the world, more studies are necessary to comprehend the genomic profiles across South America, including Colombia, considered to be the second country with the highest number of Cryptococcosis. Here, we sequenced and analyzed the genomic architecture of 29 Colombian C. neoformans isolates and evaluated the phylogenetic relationship of these strains with publicly available C. neoformans genomes. The phylogenomic analysis showed that 97% of the isolates belonged to the VNI molecular type and the presence of sub-lineages and sub-clades. We evidenced a karyotype without changes, a low number of genes with copy number variations, and a moderate number of single-nucleotide polymorphisms (SNPs). Additionally, a difference in the number of SNPs between the sub-lineages/sub-clades was observed; some were involved in crucial fungi biological processes. Our study demonstrated the intraspecific divergence of C. neoformans in Colombia. These findings provide evidence that Colombian C. neoformans isolates do not probably require significant structural changes as adaptation mechanisms to the host. To the best of our knowledge, this is the first study to report the whole genome sequence of Colombian C. neoformans isolates.

Deciphering the Association among Pathogenicity, Production and Polymorphisms of Capsule/Melanin in Clinical Isolates of Cryptococcus neoformans var. grubii VNI.

BACKGROUND: Cryptococcus neoformans is an opportunistic fungal pathogen that can cause meningitis in immunocompromised individuals. The objective of this work was to study the relationship between the phenotypes and genotypes of isolates of clinical origin from different cities in Colombia. METHODS: Genome classification of 29 clinical isolates of C. neoformans var. grubii was performed using multilocus sequence typing (MLST), and genomic sequencing was used to genotype protein-coding genes. Pathogenicity was assessed in a larval model, and melanin production and capsule size were evaluated in vitro and in vivo. RESULTS: Eleven MLST sequence types (STs) were found, the most frequent being ST69 (n = 9), ST2, ST93, and ST377 (each with n = 4). In the 29 isolates, different levels of pigmentation, capsule size and pathogenicity were observed. Isolates classified as highly pathogenic showed a tendency to exhibit larger increases in capsule size. In the analysis of polymorphisms, 48 non-synonymous variants located in the predicted functional domains of 39 genes were found to be associated with capsule size change, melanin, or pathogenicity. CONCLUSIONS: No clear patterns were found in the analysis of the phenotype and genotype of Cryptococcus. However, the data suggest that the increase in capsule size is a key variable for the differentiation of pathogenic isolates, regardless of the method used for its induction.

The Combination of Iron and Copper Increases Pathogenicity and Induces Proteins Related to the Main Virulence Factors in Clinical Isolates of Cryptococcus neoformans var. grubii.

In fungi, metals are associated with the expression of virulence factors. However, it is unclear whether the uptake of metals affects their pathogenicity. This study aimed to evaluate the effect of iron/copper in modulating pathogenicity and proteomic response in two clinical isolates of C. neoformans with high and low pathogenicity. METHODS: In both isolates, the effect of 50 µM iron and 500 µM copper on pathogenicity, capsule induction, and melanin production was evaluated. We then performed a quantitative proteomic analysis of cytoplasmic extracts exposed to that combination. Finally, the effect on pathogenicity by iron and copper was evaluated in eight additional isolates. RESULTS: In both isolates, the combination of iron and copper increased pathogenicity, capsule size, and melanin production. Regarding proteomic data, proteins with increased levels after iron and copper exposure were related to biological processes such as cell stress, vesicular traffic (Ap1, Vps35), cell wall structure (Och1, Ccr4, Gsk3), melanin biosynthesis (Hem15, Mln2), DNA repair (Chk1), protein transport (Mms2), SUMOylation (Uba2), and mitochondrial transport (Atm1). Increased pathogenicity by exposure to metal combination was also confirmed in 90% of the eight isolates. CONCLUSIONS: The combination of these metals enhances pathogenicity and increases the abundance of proteins related to the main virulence factors.

Multilocus sequence typing (MLST) of clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii in six departments of Colombia reveals high genetic diversity.

INTRODUCTION: The average annual incidence of cryptococcosis in Colombia is 0.23 cases per 100,000 inhabitants in the general population, and 1.1 cases per 1000 in inhabitants with Acquired Immune Deficiency Syndrome (AIDS). In addition, the causal fungus has been isolated from the environment, with serotypes A-B and C in different regions. This study aims to determine the genetic association between clinical and environmental isolates of C. neoformans/C. gattii in Colombia. METHODS: Multilocus sequence typing (MLST) was used to identify possible clones, providing information about the epidemiology, ecology, and etiology of this pathogen in Colombia. RESULTS: A total of 110 strains, both clinical (n=61) and environmental (n=49), with 21 MLST sequence types (ST) of C. neoformans (n=14STs) and C. gattii (n=7STs) were identified. The STs which shared clinical and environmental isolate sources were grouped in different geographical categories; for C. neoformans, ST93 was identified in six departments, ST77 in five departments; and for C. gattii, ST25 was identified in three departments and ST79 in two. CONCLUSIONS: High genetic diversity was found in isolates of C. neoformans/gattii by MLST, suggesting the presence of environmental sources harboring strains which may be sources of infection for humans, especially in immunocompromised patients; these data contribute to the information available in the country on the distribution and molecular variability of C. neoformans and C. gattii isolates recovered in Colombia.

Development and Validation of an in-House Library of Colombian Candida auris Strains with MALDI-TOF MS to Improve Yeast Identification.

: Background: Candida auris is characterized for having a high genetic variability among species. MALDI-TOF MS library contains spectra from only three strains of C. auris, which makes difficult the identification process and gives low scores at the species level. Our aim was to construct and validate an internal library to improve C. auris identification with Colombian clinical strains. METHODS: From 30 clinical strains, 770 mass spectra were obtained for the construction of the database. The validation was performed with 300 strains to compare the identification results in the BDAL and C. auris Colombia libraries. RESULTS: Our library allowed a complete, 100% identification of the evaluated strains and a significant improvement in the scores obtained, showing a better performance compared to the Bruker BDAL library. CONCLUSIONS: The strengthening of the database is a great opportunity to improve the scoring and C. auris identification. Library data are available via ProteomeXchange with identifier PXD016387.

Persistence of Clonal Azole-Resistant Isolates of Candida albicans from a Patient with Chronic Mucocutaneous Candidiasis in Colombia.

PURPOSE: The present article describes retrospectively a case of a patient with chronic mucocutaneous candidiasis (CMC) who presented recurrent Candida albicans infection since he was 6 months old. We obtained 16 isolates recovered during a 4-year period. Our purpose was to determinate the susceptibility, genotyping, and the pathogenicity profile in all the isolates. METHODS: Sixteen C. albicans were isolated from a 25-year-old male with several recurrent fungal infections admitted to Hospital. The isolates were recovered during 4 years from a different anatomical origin. We typified them by multilocus sequence typing, also we evaluated susceptibility to fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, caspofungin, and amphotericin B by microdilution method and we also test the pathogenic capacity in the Galleria mellonella model. RESULTS: Genotyping of all clinical isolates showed the persistence of the same diploid sequence type (DST). Isolates changed their susceptibility profile over time, but there were no significant statistical differences in pathogenicity. CONCLUSION: Herein, a persistent clonal isolates of C. albicans (DST 918) in a patient with CMC, showed changes in its susceptibility profile after several antifungal treatments acquiring gradual resistance to the azole drugs, which did not affect their pathogenicity.

Multilocus sequence typing (MLST) of clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii in six departments of Colombia reveals high genetic diversity

Abstract INTRODUCTION: The average annual incidence of cryptococcosis in Colombia is 0.23 cases per 100,000 inhabitants in the general population, and 1.1 cases per 1000 in inhabitants with Acquired Immune Deficiency Syndrome (AIDS). In addition, the causal fungus has been isolated from the environment, with serotypes A-B and C in different regions. This study aims to determine the genetic association between clinical and environmental isolates of C. neoformans/C. gattii in Colombia. METHODS: Multilocus sequence typing (MLST) was used to identify possible clones, providing information about the epidemiology, ecology, and etiology of this pathogen in Colombia. RESULTS: A total of 110 strains, both clinical (n=61) and environmental (n=49), with 21 MLST sequence types (ST) of C. neoformans (n=14STs) and C. gattii (n=7STs) were identified. The STs which shared clinical and environmental isolate sources were grouped in different geographical categories; for C. neoformans, ST93 was identified in six departments, ST77 in five departments; and for C. gattii, ST25 was identified in three departments and ST79 in two. CONCLUSIONS: High genetic diversity was found in isolates of C. neoformans/gattii by MLST, suggesting the presence of environmental sources harboring strains which may be sources of infection for humans, especially in immunocompromised patients; these data contribute to the information available in the country on the distribution and molecular variability of C. neoformans and C. gattii isolates recovered in Colombia.

Hospital-Associated Multicenter Outbreak of Emerging Fungus Candida auris, Colombia, 2016.

Candida auris is an emerging multidrug-resistant fungus that causes hospital-associated outbreaks of invasive infections with high death rates. During 2015-2016, health authorities in Colombia detected an outbreak of C. auris. We conducted an investigation to characterize the epidemiology, transmission mechanisms, and reservoirs of this organism. We investigated 4 hospitals with confirmed cases of C. auris candidemia in 3 cities in Colombia. We abstracted medical records and collected swabs from contemporaneously hospitalized patients to assess for skin colonization. We identified 40 cases; median patient age was 23 years (IQR 4 months-56 years). Twelve (30%) patients were <1 year of age, and 24 (60%) were male. The 30-day mortality was 43%. Cases clustered in time and location; axilla and groin were the most commonly colonized sites. Temporal and spatial clustering of cases and skin colonization suggest person-to-person transmission of C. auris. These cases highlight the importance of adherence to infection control recommendations.

Genotypic Diversity Is Independent of Pathogenicity in Colombian Strains of Cryptococcus neoformans and Cryptococcus gattii in Galleria mellonella.

Cryptococcosis is a potentially fatal opportunistic mycosis that affects the lungs and central nervous system. It has been suggested that certain strains of C. neoformans/C. gattii may have the potential to be more virulent according to the molecular type. This study aims to investigate the association between virulence in the G. mellonella model and genotypic diversity of Colombian clinical and environmental isolates of C. neoformans/C. gattii. A total of 33 clinical and 12 environmental isolates were selected according to their geographical origin and sequence types (STs). Pathogenicity was determined using the G. mellonella model, and the cell and capsular size before and after inoculation was determined. For C. neoformans, virulence in G. mellonella revealed that death occurred on average on day 6 (p < 0.05) and that ST5C, 6C, 25C and 71C were the most virulent. In C. gattii, death occurred at 7.3 days (p < 0.05), and ST47C, 58C, 75A and 106C were the most virulent. Capsular size increased for both species after passage in G. mellonella. In conclusion, the pathogenicity of Cryptococcus strains in the G. mellonella invertebrate model is independent of molecular type or pathogenicity factor, even within the same ST, but it is possible to find variable degrees of pathogenicity.

Report on novel environmental niches for Cryptococcus neoformans and Cryptococcus gattii in Colombia: Tabebuia guayacan and Roystonea regia.

Knowledge of the environmental distribution of C. neoformans/C. gattii is important in the epidemiology and ecology of the etiological agent, which causes cryptococcosis, a deadly disease worldwide. The aim of this report is to describe the presence of C. neoformans/C. gattii in new environmental niches in Colombia. A total of 837 environmental samples were collected from six different species of trees across four cities; molecular type was determined by PCR fingerprinting and RFLP. Molecular type VNI and VGIII were isolated from different species of trees, resulting in two novel niches for this pathogen: Tabebuia guayacan and Roystonea regia.

Distribution and association between environmental and clinical isolates of Cryptococcus neoformans in Bogotá-Colombia, 2012-2015.

The propagules of the fungal species Cryptococcus neoformans and C. gattii, whose varieties are distributed world wide, are the primary cause of cryptococcosis, a life threatening disease. The study of environmental and clinical isolates of Cryptococcosis is an important contribution to the epidemiology and ecology of the fungus. The aim of this work was to determine the presence of C. neoformans and C. gattii in the environment in Bogotá, Colombia's capital city and to establish the relation between clinical and environmental isolates in the period 2012-2015. From a total of 4.116 environmental samples collected between October 2012 - March 2014, 35 were positive for C. neoformans var. grubii. From 55 cryptococcosis cases reported in Bogotá during 2012-2015, 49 isolates were recovered. From those, 94% were identified as C. neoformans var. grubii molecular type VNI; 4% as VNII and 1,2% as C. neoformans var neoformans VNIV. The 84 detected clinical and environmental isolates studied had a similarity between 49-100% according with molecular typing. The correlation between environmental and clinical samples confirms the hypothesis that patients acquire the disease from environmental exposure to the fungal propagules.

Distribution and association between environmental and clinical isolates of Cryptococcus neoformans in Bogotá-Colombia, 2012-2015

The propagules of the fungal species Cryptococcus neoformans and C. gattii, whose varieties are distributed world wide, are the primary cause of cryptococcosis, a life threatening disease. The study of environmental and clinical isolates of Cryptococcosis is an important contribution to the epidemiology and ecology of the fungus. The aim of this work was to determine the presence of C. neoformans and C. gattii in the environment in Bogotá, Colombia’s capital city and to establish the relation between clinical and environmental isolates in the period 2012-2015. From a total of 4.116 environmental samples collected between October 2012 - March 2014, 35 were positive for C. neoformans var. grubii. From 55 cryptococcosis cases reported in Bogotá during 2012-2015, 49 isolates were recovered. From those, 94% were identified as C. neoformans var. grubii molecular type VNI; 4% as VNII and 1,2% as C. neoformans var neoformans VNIV. The 84 detected clinical and environmental isolates studied had a similarity between 49-100% according with molecular typing. The correlation between environmental and clinical samples confirms the hypothesis that patients acquire the disease from environmental exposure to the fungal propagules.

Caracterización molecular de aislamientos de Shigella sonnei recuperados en el programa de vigilancia por el laboratorio de la enfermedad diarreica aguda en Colombia / Molecular characterization of Shigella sonnei isolates recovered by the Laboratory Surveillance Program for Acute Diarrheal Disease in Colombia

Introducción. En Colombia, Shigella sonnei es uno de los serotipos más frecuentemente aislados (53,4 %) de muestras clínicas humanas asociadas a la enfermedad diarreica aguda. La identificación de patrones de restricción del ADN mediante electroforesis en gel de campo pulsado constituye la base de la vigilancia molecular de S. sonnei . Objetivo. Establecer la base de la vigilancia molecular de S. sonnei en Colombia mediante electroforesis en gel de campo pulsado. Materiales y métodos. Se estudiaron 102 de los 2.048 aislamientos de S. sonnei remitidos por la Red Nacional de Laboratorios entre 1997 y marzo del 2013; la selección se hizo de acuerdo con el patrón de resistencia antimicrobiana, el origen de la muestra y la relación con brotes. Se determinó el patrón genético mediante electroforesis en gel de campo pulsado con las enzimas de restricción XbaI y Blnl, según el protocolo de la red PulseNet International. El análisis de los patrones electroforéticos se hizo con el programa GelCompar II, versión 4.0. Resultados. Se obtuvieron 42 patrones electroforéticos con una similitud de 70 a 100 %. El patrón más frecuente fue COIN08J16X01.0017 (17,6 %), seguido por los patrones COIN04J16X01.0004 (9,8 %) y COIN02J16X01.0002 (5,8 %), y el 66,8 % restante se asoció con otros patrones electroforéticos. El análisis de brotes demostró la relación genética de cada brote con 100 % de similitud en la identificación; el patrón más frecuente en los brotes fue el COIN08J16X01.0017 (17,1 %). Conclusión. Se estableció la base de datos genotípicos de aislamientos de S. sonnei a nivel nacional mediante electroforesis en gel de campo pulsado; se incluyeron los 42 patrones únicos identificados en este estudio.

Introduction: In Colombia, Shigella sonnei is one of the most frequently isolated serotypes (53.4%) in human clinical samples associated with diarrheal acute disease. The identification of DNA restriction patterns by pulsed field gel electrophoresis is the basis for the molecular surveillance of S. sonnei . Objective: To establish the basis for the molecular surveillance of S. sonnei in Colombia using pulsed-field gel electrophoresis. Materials and methods: We studied 102 of 2,048 S. sonnei isolates referred by the National Laboratory Network between 1997 and March, 2013; the selection was made according to the antimicrobial multiresistance profile, the source of samples, and the relation to outbreaks. The genetic profile was determined by pulsed field gel electrophoresis using the restriction enzymes XbaI and BlnI in accordance with the PulseNet International protocol. The electrophoretic patterns were analyzed with the GelCompare II, version 4.0 software. Results: We obtained 42 electrophoretic patterns with a 70% to 100% similarity. The most frequent pattern was COIN08J16X01.0017 with 17.6%, followed by patterns COIN04J16X01.0004 with 9.8%, and COIN02J16X01.0002 with 5.8%, while the remaining 66.8% was associated with other electrophoretic patterns. The analysis of 10 outbreaks demonstrated their genetic relation with a 100% of similarity; the most frequent pattern in outbreaks was COIN08J16X01.0017 with 17.1%. Conclusion: The genotypic database for Shigella sonnei isolates was established using pulsed field gel electrophoresis including the 42 unique patterns identified in this study.

[Molecular characterization of Shigella sonnei isolates recovered by the Laboratory Surveillance Program for Acute Diarrheal Disease in Colombia]. / Caracterización molecular de aislamientos de Shigella sonnei recuperados en el programa de vigilancia por el laboratorio de la enfermedad diarreica aguda en Colombia.

INTRODUCTION: In Colombia, Shigella sonnei is one of the most frequently isolated serotypes (53.4%) in human clinical samples associated with diarrheal acute disease. The identification of DNA restriction patterns by pulsed field gel electrophoresis is the basis for the molecular surveillance of S. sonnei . OBJECTIVE: To establish the basis for the molecular surveillance of S. sonnei in Colombia using pulsed-field gel electrophoresis. MATERIALS AND METHODS: We studied 102 of 2,048 S. sonnei isolates referred by the National Laboratory Network between 1997 and March, 2013; the selection was made according to the antimicrobial multiresistance profile, the source of samples, and the relation to outbreaks. The genetic profile was determined by pulsed field gel electrophoresis using the restriction enzymes XbaI and BlnI in accordance with the PulseNet International protocol. The electrophoretic patterns were analyzed with the GelCompare II, version 4.0 software. RESULTS: We obtained 42 electrophoretic patterns with a 70% to 100% similarity. The most frequent pattern was COIN08J16X01.0017 with 17.6%, followed by patterns COIN04J16X01.0004 with 9.8%, and COIN02J16X01.0002 with 5.8%, while the remaining 66.8% was associated with other electrophoretic patterns. The analysis of 10 outbreaks demonstrated their genetic relation with a 100% of similarity; the most frequent pattern in outbreaks was COIN08J16X01.0017 with 17.1%. CONCLUSION: The genotypic database for Shigella sonnei isolates was established using pulsed field gel electrophoresis including the 42 unique patterns identified in this study.

The Fungus among Us: Cryptococcus neoformans and Cryptococcus gattii Ecological Modeling for Colombia.

The environmental isolation of Cryptococcus spp. is typically a difficult undertaking. Collecting samples in the field is costly in terms of travel, personnel time and materials. Furthermore, the recovery rate of Cryptococcus spp. may be very low, thereby requiring a large number of samples to be taken without any guarantee of success. Ecological niche modeling is a tool that has traditionally been used to forecast the distribution of plant and animal of species for biodiversity and conservation purposes. Here, we use it in a public health application to produce risk area maps for cryptococcal disease in Colombia. The Genetic Algorithm for Ruleset Production (GARP) was used to create models for Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii), based on environmental sampling and clinical records data recorded since 1987. These maps could be used to focus public health messaging related to cryptococcal disease, and it enables us to characterize the ecological niche for Cryptococcus in Colombia. We found that the OPEN ACCESS J. Fungi 2015, 1 333 ecological niche for C. gattii in Colombia is quite diverse, establishing itself in sub-tropical and temperate ecoregions within the country. This suggests that C. gattii is highly adaptive to different ecological conditions in Colombia and different regions of the world.