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.
Abstract
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 (β-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.
•Candida auris is an emerging difficult-to-manage multidrug-resistant yeast.•Most C. auris isolates were azole-resistant but susceptible to echinocandins.•Agreement between the two reference ...antifungal susceptibility testing methods was 90%.•Antifungal susceptibility results should be interpreted with care if VITEK®2 is used.
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.
Abstract
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.
In this review, the authors illustrate the biological and clinical aspects of using phage therapy, with a focus on the CRISPR-Cas mechanism, and the interesting activity of anti-CRISPR proteins.
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.
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.
•We applied MALDI-TOF directly to blood cultures to identify bloodstream infections (BSIs).•MALDI-TOF allows earlier identification of BSIs and facilitates treatment management.•Our protocol requires ...few steps and has an easily made buffer.•Allows better use of robust MALDI TOF technology, with results available in minutes.•MALDI-TOF MS is a fast and cheap method for the identification of bacteria.
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.
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.
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).
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.
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.
Background:
is characterized for having a high genetic variability among species. MALDI-TOF MS library contains spectra from only three strains of
, 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
identification with Colombian clinical strains.
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
Colombia libraries.
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.
The strengthening of the database is a great opportunity to improve the scoring and
identification. Library data are available via ProteomeXchange with identifier PXD016387.
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.
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%).
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.
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