Applying targeted gene hybridization capture to viruses with a focus to SARS-CoV-2.

Although next-generation sequencing technologies are advancing rapidly, many research topics often require selective sequencing of genomic regions of interest. In addition, sequencing low-titre viruses is challenging, especially for coronaviruses, which are the largest RNA viruses. Prior to sequencing, enrichment of viral particles can help to significantly increase target sequence information as well as avoid large sequencing efforts and, consequently, can increase sensitivity and reduce sequencing costs. Targeting nucleic acids using capture by hybridization is another efficient method that can be performed by applying complementary probes (DNA or RNA baits) to directly enrich genetic information of interest while removing background non-target material. In studies where sequence capture by hybridization has been applied to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, most authors agree that this technique is useful to easily access sequence targets in complex samples. Furthermore, this approach allows for complete or near-complete sequencing of the viral genome, even in samples with low viral load or poor nucleic acid integrity. In addition, this strategy is highly efficient at discovering new variants by facilitating downstream investigations, such as phylogenetics, epidemiology, and evolution. Commercial kits, as well as in-house protocols, have been developed for enrichment of viral sequences. However, these kits have multiple variations in procedure, with differences in performance. This review compiles and describes studies in which hybridization capture has been applied to SARS-CoV-2 variant genomes.

Antimicrobial Resistance Profiles of Staphylococcus Isolated from Cows with Subclinical Mastitis: Do Strains from the Environment and from Humans Contribute to the Dissemination of Resistance among Bacteria on Dairy Farms in Colombia?

Staphylococcus is a very prevalent etiologic agent of bovine mastitis, and antibiotic resistance contributes to the successful colonization and dissemination of these bacteria in different environments and hosts on dairy farms. This study aimed to identify the antimicrobial resistance (AMR) genotypes and phenotypes of Staphylococcus spp. isolates from different sources on dairy farms and their relationship with the use of antibiotics. An antimicrobial susceptibility test was performed on 349 Staphylococcus strains (S. aureus, n = 152; non-aureus staphylococci (NAS), n = 197) isolated from quarter milk samples (QMSs) from cows with subclinical mastitis (176), the teats of cows (116), the milking parlor environment (32), and the nasal cavities of milk workers (25). Resistance and multidrug resistance percentages found for S. aureus and NAS were (S. aureus = 63.2%, NAS = 55.8%) and (S. aureus = 4.6%, NAS = 11.7%), respectively. S. aureus and NAS isolates showed resistance mainly to penicillin (10 IU) (54.1% and 32.4%) and ampicillin (10 mg) (50.3% and 27.0%) drugs. The prevalence of AMR Staphylococcus was higher in environmental samples (81.3%) compared to other sources (52.6-76.0%). In S. aureus isolates, the identification of the blaZ (83.9%), aacAaphD (48.6%), ermC (23.5%), tetK (12.9%), and mecA (12.1%) genes did not entirely agree with the AMR phenotype. We conclude that the use of ß-lactam antibiotics influences the expression of AMR in Staphylococcus circulating on dairy farms and that S. aureus isolates from the environment and humans may be reservoirs of AMR for other bacteria on dairy farms.

Proteomics and Genetic Approaches Elucidate the Circulation of Low Variability Staphylococcus aureus Strains on Colombian Dairy Farms.

Staphylococcus aureus is one of the most prevalent pathogens causing bovine mastitis in the world, in part because of its ease of adaptation to various hosts and the environment. This study aimed to determine the prevalence of S. aureus in Colombian dairy farms and its relationship with the causal network of subclinical mastitis. From thirteen dairy farms enrolled, 1288 quarter milk samples (QMS) and 330 teat samples were taken from cows with positive (70.1%) and negative California Mastitis Test (CMT). In addition, 126 samples from the milking parlor environment and 40 from workers (nasal) were collected. On each dairy farm, a survey was conducted, and the milking process was monitored on the day of sampling. S. aureus was identified in 176 samples, i.e., 138 QMS, 20 from teats, 8 from the milking parlor environment, and 10 from workers' nasal swabs. Isolates identified as S. aureus underwent proteomics (clustering of mass spectrum) and molecular (tuf, coa, spa Ig, clfA, and eno genes) analysis. Regarding proteomics results, isolates were distributed into three clusters, each with members from all sources and all farms. Concerning molecular analysis, the virulence-related genes clfA and eno were identified in 41.3% and 37.8% of S. aureus isolates, respectively. We provide evidence on the circulation of S. aureus strains with limited variability among animals, humans, and the environment. The parameters with the lowest compliance in the farms which may be implicated in the transmission of S. aureus are the lack of handwashing and abnormal milk handling.

The Mortality Attributable to Candidemia in C. auris Is Higher than That in Other Candida Species: Myth or Reality?

Candida auris has become a major health threat due to its transmissibility, multidrug resistance and severe outcomes. In a case-control design, 74 hospitalised patients with candidemia were enrolled. In total, 22 cases (29.7%) and 52 controls (C. albicans, 21.6%; C. parapsilosis, 21.6%; C. tropicalis, 21.6%; C. glabrata, 1.4%) were included and analysed in this study. Risk factors, clinical and microbiological characteristics and outcomes of patients with C. auris and non-auris Candida species (NACS) candidemia were compared. Previous fluconazole exposure was significantly higher in C. auris candidemia patients (OR 3.3; 1.15-9.5). Most C. auris isolates were resistant to fluconazole (86.3%) and amphotericin B (59%) whilst NACS isolates were generally susceptible. No isolates resistant to echinocandins were detected. The average time to start antifungal therapy was 3.6 days. Sixty-three (85.1%) patients received adequate antifungal therapy, without significant differences between the two groups. The crude mortality at 30 and 90 days of candidemia was up to 37.8% and 40.5%, respectively. However, there was no difference in mortality both at 30 and 90 days between the group with candidemia by C. auris (31.8%) and by NACS (42.3%) (OR 0.6; 95% IC 0.24-1.97) and 36.4% and 42.3% (0.77; 0.27-2.1), respectively. In this study, mortality due to candidemia between C. auris and NACS was similar. Appropriate antifungal therapy in both groups may have contributed to finding no differences in outcomes.

Emergence and circulation of azole-resistant C. albicans, C. auris and C. parapsilosis bloodstream isolates carrying Y132F, K143R or T220L Erg11p substitutions in Colombia.

Methods: Over a four-year period, 123 Candida bloodstream isolates were collected at a quaternary care hospital. The isolates were identified by MALDI-TOF MS and their fluconazole (FLC) susceptibility patterns were assessed according to CLSI guidelines. Subsequently, sequencing of ERG11, TAC1 or MRR1, and efflux pump activity were performed for resistant isolates. Results: Out of 123 clinical strains,C. albicans accounted for 37.4%, followed by C. tropicalis 26.8%, C. parapsilosis 19.5%, C. auris 8.1%, C. glabrata 4.1%, C. krusei 2.4% and C. lusitaniae 1.6%. Resistance to FLC reached 18%; in addition, a high proportion of isolates were cross-resistant to voriconazole. Erg11 amino acid substitutions associated with FLC-resistance (Y132F, K143R, or T220L) were found in 11/19 (58%) of FLCresistant isolates. Furthermore, novel mutations were found in all genes evaluated. Regarding efflux pumps, 8/19 (42%) of FLC-resistant Candida spp strains showed significant efflux activity. Finally, 6/19 (31%) of FLC-resistant isolates neither harbored resistance-associated mutations nor showed efflux pump activity. Among FLC-resistant species, C. auris 7/10 (70%) and C. parapsilosis 6/24 (25%) displayed the highest percentages of resistance (C. albicans 6/46, 13%). Discussion: Overall, 68% of FLC-resistant isolates exhibited a mechanism that could explain their phenotype (e.g. mutations, efflux pump activity, or both). We provide evidence that isolates from patients admitted to a Colombian hospital harbor amino acid substitutions related to resistance to one of the most commonly used molecules in the hospital setting, with Y132F being the most frequently detected.

Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II: Antifungal Activity against Reference Strains and Clinical Isolates of Candida spp.

Antimicrobial peptides (AMPs) are considered to be a valuable source for the identification and/or design of promising candidates for the development of antifungal treatments, since they have advantages such as lower tendency to induce resistance, ease of production, and high purity and safety. Bovine lactoferricin (LfcinB) and buforin II (BFII) are AMPs to which great antimicrobial potential has been attributed. The minimum motives with antimicrobial activity derived from LfcinB and BFII are RRWQWR and RLLR, respectively. Nine chimeras containing the minimum motives of both peptides were synthesized and their antifungal activity against fluconazole (FLC)-sensitive and resistant C. albicans, C. glabrata, and C. auris strains was evaluated. The results showed that peptides C9: (RRWQWR)2K-Ahx-RLLRRRLLR and C6: KKWQWK-Ahx-RLLRRLLR exhibited the greatest antifungal activity against two strains of C. albicans, a FLC-sensitive reference strain and a FLC-resistant clinical isolate; no medically significant results were observed with the other chimeras evaluated (MIC ~200 µg/mL). The chimera C6 was also active against sensitive and resistant strains of C. glabrata and C. auris. The combination of branched polyvalent chimeras together with FLC showed a synergistic effect against C. albicans. In addition to exhibiting antifungal activity against reference strains and clinical isolates of Candida spp., they also showed antibacterial activity against both Gram-positive and Gram-negative bacteria, suggesting that these chimeras exhibit a broad antimicrobial spectrum and can be considered to be promising molecules for therapeutic applications.

Phages, anti-CRISPR proteins, and drug-resistant bacteria: what do we know about this triad?

Phages are viruses that infect bacteria, relying on their genetic machinery to replicate. To survive the constant attack of phages, bacteria have developed diverse defense strategies to act against them. Nevertheless, phages rapidly co-evolve to overcome these barriers, resulting in a constant, and often surprising, molecular arms race. Thus, some phages have evolved protein inhibitors known as anti-CRISPRs (∼50-150 amino acids), which antagonize the bacterial CRISPR-Cas immune response. To date, around 45 anti-CRISPRs proteins with different mechanisms and structures have been discovered against the CRISPR-Cas type I and type II present in important animal and human pathogens such as Escherichia, Morganella, Klebsiella, Enterococcus, Pseudomonas, Staphylococcus, and Salmonella. Considering the alarming growth of antibiotic resistance, phage therapy, either alone or in combination with antibiotics, appears to be a promising alternative for the treatment of many bacterial infections. In this review, we illustrated the biological and clinical aspects of using phage therapy; furthermore, the CRISPR-Cas mechanism, and the interesting activity of anti-CRISPR proteins as a possible weapon to combat bacteria.

Evaluation of Anti-Candida Potential of Piper nigrum Extract in Inhibiting Growth, Yeast-Hyphal Transition, Virulent Enzymes, and Biofilm Formation.

Due to the increased incidence of fungal infections and the emergence of antifungal resistance mainly by Candida species, the need for safe and effective novel therapies is imperative. Consequently, plants and herbs are a powerful source to combat infections. Here, we evaluated the anti-Candida potential of an ethanolic extract from Piper nigrum. The phytochemical analysis of P. nigrum revealed bioactive compounds such as alkaloids, terpenoids, and tannis. Our results showed that P. nigrum extract suppressed the virulence factors of C. albicans strains, including hyphae formation in both liquid and solid media, reduced secretion of phospholipases/proteinases, and affected biofilm formation. Furthermore, the P. nigrum extract showed no hemolytic effect in vitro and exhibited reduced cytotoxicity on Vero cells and G. mellonella larvae at concentrations that inhibited hyphae and biofilm in C. albicans. Moreover, the extract demonstrated antifungal activity against C. auris strains. In conclusion, the P. nigrum extract affected the growth and morphogenesis of Candida (even in resistant strains), demonstrating that this plant has an anti-candida activity and represents a promising resource for discovering novel antifungal compounds.

Prototheca Infections and Ecology from a One Health Perspective.

Prototheca microalgae were only recognized as pathogens of both humans and animals in the 1960s; however, since then, these microbes have been drawing increasing interest in both human and veterinary medicine. The first human outbreak of protothecosis in a tertiary care chemotherapy ward in 2018 further highlighted the need to understand in more depth and detail their ecology, etiology, pathogenesis and routes of transmission between different hosts, environments and habitats from a One Health perspective. Protothecal infections have been reported in a growing number of cattle herds around the world in recent decades, and Prototheca has become an important bovine mastitis pathogen in certain countries and regions. The survival of Prototheca in the environment and its ability to spread in the herd pose a serious challenge to the management of infected dairy farms. Prevention of the disease is particularly important, as there is no effective and reliable treatment for it and the chances of self-healing are minimal. Therefore, the development of more effective drugs is needed for the treatment of human and animal protothecosis. The prudent use of antibiotics and their replacement by alternative or preventive measures, when possible, may further contribute to the control of protothecal infections.

Head-to-head comparison of CLSI, EUCAST, Etest and VITEK®2 results for Candida auris susceptibility testing.

The susceptibility of 31 Candida auris clinical isolates was evaluated by four methods, namely the microdilution reference method according to Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines as well as Etest and VITEK®2. Essential agreement between the two reference methods was 90%. Etest showed a better overall agreement with the reference methods (94% and 81% for CLSI and EUCAST, respectively) than VITEK®2 (70% and 72%, respectively). Discrepancies were found for fluconazole (FLC) and amphotericin B. Considering categorical agreement (CDC tentative breakpoints), the majority of isolates were considered FLC-resistant (93.6% and 80.6% by CLSI and EUCAST, respectively). Furthermore, all isolates were considered susceptible to echinocandins by all methods. Susceptibility results should be interpreted with care if the VITEK®2 system is used to guide therapeutic decisions for C. auris infections.

CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata.

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.

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.

Genotypic, proteomic, and phenotypic approaches to decipher the response to caspofungin and calcineurin inhibitors in clinical isolates of echinocandin-resistant Candida glabrata.

BACKGROUND: Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets. OBJECTIVES: We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates. METHODS: Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival. RESULTS: F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin-calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity. CONCLUSIONS: Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.

Robust, Comprehensive Molecular, and Phenotypical Characterisation of Atypical Candida albicans Clinical Isolates From Bogotá, Colombia.

Candida albicans is commensal in human microbiota and is known to be the commonest opportunistic pathogen, having variable clinical outcomes that can lead to up to 60% mortality. Such wide clinical behaviour can be attributed to its phenotypical plasticity and high genetic diversity. This study characterised 10 Colombian clinical isolates which had already been identified as C. albicans by molecular tests; however, previous bioinformatics analysis of protein mass spectra and phenotypical characteristics has shown that this group of isolates has atypical behaviour, sharing characteristics of both C. africana and C. albicans. This study was aimed at evaluating atypical isolates' pathogenic capability in the Galleria mellonella model; susceptibility profiles were determined and MLST was used for molecular characterisation. Cluster analysis, enabling unbiased bootstrap to classify the isolates and establish their cluster membership and e-BURST, was used for establishing clonal complexes (CC). Both approaches involved using representative MLST data from the 18 traditional C. albicans clades, as well as C. albicans-associated and minor species. Ten atypical isolates were distributed as follows: 6/10 (B71, B41, B60, R6, R41, and R282) were grouped into a statistically well-supported atypical cluster (AC) and constituted a differentiated CC 6; 2/10 of the isolates were clearly grouped in clade 1 and were concurrent in CC 4 (B80, B44). Another 2/10 atypical isolates were grouped in clade 10 and concurred in CC 7 (R425, R111); most atypical isolates were related to geographically distant isolates and some represented new ST. Isolates B41 and R41 in the AC had greater virulence. Isolate B44 was fluconazole-resistant and was grouped in clade 1. The atypical nature of the isolates studied here was demonstrated by the contrast between phenotypical traits (C. africana-like), molecular markers (C. albicans-like), virulence, and antifungal resistance, highlighting the widely described genetic plasticity for this genus. Our results showed that the atypical isolates forming well-differentiated groups belonged to C. albicans. Our findings could contribute towards developing molecular epidemiology approaches for managing hospital-acquired infection.

In-house protocol and performance of MALDI-TOF MS in the early diagnosis of bloodstream infections in a fourth-level hospital in Colombia: Jumping to full use of this technology.

BACKGROUND: Bloodstream infections (BSIs) are a major cause of mortality in hospitalized patients. Rapid diagnosis is crucial because any delay in the antimicrobial treatment is associated with an increase in adverse patient outcomes. The application of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technology directly to blood cultures permits earlier identification of BSIs and facilitates treatment management. METHODS: A total of 470 positive blood cultures from patient samples were analyzed using Standard Aerobic/F and Anaerobic/F blood culture media. Isolates were identified using conventional identification methods and by the direct method using the MALDI-TOF MS system. RESULTS: In 470 blood cultures, the direct method showed good identification results (420/470, 89%); specifically, accurate species and genus identification in 283/470 (60%), and only correct genus identification in 137/470 (29%). The direct protocol had better performance for Gram-negative compared to Gram-positive bacteria (97% vs 76%) and was unable to identify the positive blood cultures for both yeasts and some bacteria, mostly Gram-positive (50/470). CONCLUSIONS: The protocol used here gave good and reliable results, being available up to 24 h earlier, while also leading to better use of MALDI-TOF.

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.

Impact of calmodulin inhibition by fluphenazine on susceptibility, biofilm formation and pathogenicity of caspofungin-resistant Candida glabrata.

BACKGROUND: In recent decades, Candida glabrata has emerged as a frequent cause of life-threatening fungal infection. In C. glabrata, echinocandin resistance is associated with mutations in FKS1/FKS2 (ß-1,3-glucan synthase). The calmodulin/calcineurin pathway is implicated in response to antifungal stress and calcineurin gene disruption specifically reverses Fks2-mediated resistance of clinical isolates. OBJECTIVES: We evaluated the impact of calmodulin inhibition by fluphenazine in two caspofungin-resistant C. glabrata isolates. METHODS: C. glabrata isolates were identified by ITS1/ITS4 (where ITS stands for internal transcribed spacer) sequencing and the echinocandin target FKS1/FKS2 genes were sequenced. Susceptibility testing of caspofungin in the presence of fluphenazine was performed by a modified CLSI microbroth dilution method. The effect of the fluphenazine/caspofungin combination on heat stress (37°C or 40°C), oxidative stress (0.2 and 0.4 mM menadione) and biofilm formation (polyurethane catheter) was analysed. A Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of this combination on larval survival. RESULTS: F659del was found in the FKS2 gene of both resistant strains. In these clinical isolates, fluphenazine increased susceptibility to caspofungin and reduced their thermotolerance. Furthermore, the fluphenazine/caspofungin combination significantly impaired biofilm formation in an in vitro polyurethane catheter model. All these features participated in the increasing survival of infected G. mellonella after combination treatment in comparison with caspofungin alone. CONCLUSIONS: In a repurposing strategy, our findings confirm that calmodulin could provide a relevant target in life-threatening fungal infectious diseases.

Cerebral phaeohyphomycosis caused by Alternaria spp.: A case report.

Phaeohyphomycosis is a group of infections caused by pigmented, black, dematiaceous fungi and is responsible for cutaneous, superficial and deep mycoses, disseminated infection and brain abscesses. The primary agents involved include Alternaria spp., Exophiala spp. and Cladophialophora spp. Invasive systemic presentation is rare and in most cases is associated with immunosuppression; for this reason, reported cases of Alternaria spp. infection are scarce. This report describes the case of a 66-year-old man with a history of renal transplantation from a cadaveric donor 1 year ago, which was considered as the primary risk factor. The characteristics of the infection, procedures performed, microbiological findings and treatment provided are described.

Comparison between MALDI-TOF MS and MicroScan in the identification of emerging and multidrug resistant yeasts in a fourth-level hospital in Bogotá, Colombia.

BACKGROUND: The introduction of MALDI-TOF MS in the clinical microbiology laboratory has modified the approaches for the identification of fungi. Thanks to this tool, it is possible to identify cryptic species, which possess critical susceptibility patterns. Clinical strains were identified using the MicroScan and MALDI-TOF MS systems. Discrepant results from both methods were investigated using ITS rDNA barcoding. Finally, these isolates were also tested for in vitro susceptibility. RESULTS: The percentage of agreement between both methods to 498 yeast isolates was of 93.6% (32 discrepant isolates). The concordance of ITS sequencing with MALDI-TOF MS was higher (99%) than that of MicroScan (94%). Several of these discordant yeasts displayed high MICs for antifungal agents. CONCLUSIONS: Our study highlights the need of the MS and molecular approaches such as MALDI-TOF MS and ITS rDNA barcoding for the correct identification of emerging or cryptic yeast species; besides, some of these could be multidrug resistant. This work was the first experience in the implementation of the MALDI-TOF MS technology in Colombia. We found the first uncommon yeasts including Candida auris and we could identify Trichosporon faecalis. Our work highlights a clear necessity of an accurate yeast identification as a much more pertinent technique than the susceptibility profiles, because the most unusual yeasts exhibit resistance profiles to the few available antifungals.