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Fungal resistance is a public health concern due to the limited availability of antifungal resources and the complexities associated with treating persistent fungal infections. Azoles are thus far the primary line of defense against fungi. Specifically, azoles inhibit the conversion of lanosterol to ergosterol, producing defective sterols and impairing fluidity in fungal plasmatic membranes. Studies on azole resistance have emphasized specific point mutations in CYP51/ERG11 proteins linked to resistance. Although very insightful, the traditional approach to studying azole resistance is time-consuming and prone to errors during meticulous alignment evaluation. It relies on a reference-based method using a specific protein sequence obtained from a wild-type (WT) phenotype. Therefore, this study introduces a machine learning (ML)-based approach utilizing molecular descriptors representing the physiochemical attributes of CYP51/ERG11 protein isoforms. This approach aims to unravel hidden patterns associated with azole resistance. The results highlight that descriptors related to amino acid composition and their combination of hydrophobicity and hydrophilicity effectively explain the slight differences between the resistant non-wild-type (NWT) and WT (nonresistant) protein sequences. This study underscores the potential of ML to unravel nuanced patterns in CYP51/ERG11 sequences, providing valuable molecular signatures that could inform future endeavors in drug development and computational screening of resistant and nonresistant fungal lineages.

Spheroids of intestinal cells (Caco-2) were used to evaluate the adhesion/invasion ability of Listeria monocytogenes (pathogen) and Lactobacillus sakei 1 (potential probiotic). Besides, transcriptomic analyses of Caco-2 cells in three dimensional cultures were done, with the aim of revealing possible host-foodborne bacteria interactions. Result of adhesion assay for L. monocytogenes in Caco-2 spheroids was 22.86 ± 0.33%, but it was stimulated in acidic pH (4.5) and by the presence of 2% sucrose (respectively, 32.56 ± 1.35% and 33.25 ± 1.26%). Conversely, the invasion rate of L. monocytogenes was lower at pH 4.5, in comparison with non-stressed controls (18.89 ± 1.05% and 58.65 ± 0.30%, respectively). L. sakei 1 adhered to Caco-2 tridimensional cell culture (27.30 ± 2.64%), with no invasiveness. There were 19 and 21 genes down and upregulated, respectively, in tridimensional Caco-2 cells, upon infection with L. monocytogenes, which involved immunity, apoptosis; cytoprotective responses, cell signalling-regulatory pathways. It was evidenced despite activation or deactivation of several pathways in intestinal cells to counteract infection, the pathogen was able to hijack many host defense mechanisms. On the other hand, the probiotic candidate L. sakei 1 was correlated with decreased transcription of two genes in Caco-2 cells, though it stimulated the expression of 14 others, with diverse roles in immunity, apoptosis, cytoprotective response and cell signalling-regulatory pathways. Our data suggest the use of tridimensional cell culture to mimic the intestinal epithelium is a good model for gathering broad information on the putative mechanisms of interaction between host and bacteria of importance for food safety, which can serve as a basis for further in-depth investigation.

For this research communication, 90 samples of a Brazilian dairy were combined into four groups (raw material, final product, food-contact and non-food contact surfaces) and analyzed by metataxonomics based on 16S rRNA gene sequencing. The results showed high alpha-diversity indexes for final product and non-food contact surfaces but, overall, beta-diversity indexes were low. The samples were separated in two main clusters, and the core microbiota was composed by Macrococcus, Alkaliphilus, Vagococcus, Lactobacillus, Marinilactibacillus, Streptococcus, Lysinibacillus, Staphylococcus, Clostridium, Halomonas, Lactococcus, Enterococcus, Bacillus and Psychrobacter. These results highlight that rare taxa occur in dairies, and this may aid the development of strategies for food protection.

Dairy foods are complex ecosystems composed of microorganisms from different origins that can affect flavor and safety of final products. The objective of this paper is to assess the in-house microbiota of two Brazilian dairies and to discuss the possible implications of the taxa determined for food protection. In total, 27 samples from dairies were cultured in selective (Baird Parker, de Man, Rogosa and Sharpe) and non-selective (Brain Heart Infusion) media, and the isolates were identified by Sanger sequencing. Moreover, metagenomic DNA was directly extracted from samples and the structure of the bacterial community was determined by massive DNA sequencing followed by bioinformatics analyses. The results showed the majority of isolates belonged to the group of lactic acid bacteria, but Enterobacteriaceae, Staphylococcacceae, Bacillaceae, Pseudomonadaceae and Moraxellaceae were also detected. From the reads obtained in metataxonomics analyses, a heatmap was constructed and the top 20 OTUs (operational taxonomic units) were determined. Besides, 12 most prevalent bacterial taxa were assigned to the core microbiota of the dairies evaluated, which included Thiomonas thermosulfata, Alkalibacillus salilacus, Pseudomonas clemancea, Erythrobacter aquimans, Tetragenococcus doogicus, Macrococcus brunensis, Pseudomonas ludensis, Streptococcus dentinousetti, Serratia entomophila, Vagococcus teuberi, Lactococcus fujiensis and Tolumonas auensis. In conclusion, the results reveal the presence of bacteria that may be related to spoilage and also foodborne diseases, in microbial niches that also present rare taxa, highlighting the importance to consider culture-independent results to evaluate and improve food safety.

ABSTRACT Sarocladium spp. are filamentous fungi commonly associated with plant diseases and only rarely cause hyalohyphomycosis in humans. Immunosuppressed patients are at risk for this infection, which typically presents with skin and subcutaneous lesions that may eventually disseminate to internal organs. This study reports a case of a man in intensive care following SARS-CoV-2 infection. During hospitalization, he developed neutropenia, persistent fever, and a cavitary lung lesion. Sarocladium spp. was isolated from blood cultures, and the patient was treated with voriconazole, leading to a successful cure. To our knowledge, this is the first reported case of Sarocladium implicatum infection in a COVID-19 patient, underscoring the importance of monitoring opportunistic fungal infections in immunocompromised individuals, particularly during epidemics and pandemics.

Microbial communities infiltrate the respiratory tract of cystic fibrosis patients, where chronic colonization and infection lead to clinical decline. This report aims to provide an overview of the diversity of bacterial and fungal species from the airway secretion of three young CF patients with severe pulmonary disease. The bacterial and fungal microbiomes were investigated by culture isolation, metataxonomics, and metagenomics shotgun. Virulence factors and antibiotic resistance genes were also explored. was isolated from cultures and identified in high incidence from patient sputum samples. sp., sp., , and were isolated sporadically. Metataxonomics and metagenomics detected fungal reads ( , , and  sp.) in one sputum sample. The main pathogenic bacteria identified were , , complex, and . The canonical core CF microbiome is composed of species from the genera , and . Thus, the airways of the three young CF patients presented dominant bacterial genera and interindividual variability in microbial community composition and diversity. Additionally, a wide diversity of virulence factors and antibiotic resistance genes were identified in the CF lung microbiomes, which may be linked to the clinical condition of the CF patients. Understanding the microbial community is crucial to improve therapy because it may have the opposite effect, restructuring the pathogenic microbiota. Future studies focusing on the influence of fungi on bacterial diversity and microbial interactions in CF microbiomes will be welcome to fulfill this huge gap of fungal influence on CF physiopathology.

Disseminated fusariosis is treated with amphotericin B and voriconazole. To determine adequate therapy, the minimal inhibitory concentration (MIC) is used. However, MIC analysis is based on visual observation and requires a long period of fungal incubation. The measure of the minimal profile change concentration (MPCC) using MALDI-TOF MS is a quick spectral method that has presented good results in determining the antimicrobial resistance of yeasts. However, there is a lack of information on filamentous fungi. In the present work, 13 Fusarium spp. clinical isolates and two reference strains were used. MIC was obtained according to the M38-A2 protocol of the Clinical Laboratory Standards Institute, while MPPC was obtained following the initial steps of the M38-A2 protocol. Both Biotyper and the Rstudio environment were used to analyze mass spectra. For some fungal strains, the data obtained from the software MALDI Biotyper Compass 4.1 led to fuzzy heatmaps resulting in difficult interpretation, while heatmaps obtained using Rstudio tools generated better MPCC resolutions. Herein, 86.6% of the AMB MPCC values were highly correlated with the gold-standard AMB MIC. MALDI-TOF MS is a prominent tool used to determine MPCCs quicker, cost-effectively, and more accurately for Fusarium spp. strains. However, better statistical analyses could help measure the technique’s limit detection.

Acinetobacter bereziniae has recently gained medical notoriety due to its emergence as a multidrug resistance and healthcare-associated pathogen. In this study, we report the whole-genome characterization of an A. bereziniae strain (A321) recovered from an infected semiaquatic turtle, as well as a comparative analysis of A. bereziniae strains circulating at the human-animal-environment interface. Strain A321 displayed a multidrug resistance profile to medically important antimicrobials, which was supported by a wide resistome. The novel Tn 5393m transposon and a qnrB19 -bearing ColE1-like plasmid were identified in A321 strain. Novel OXA-229-like β-lactamases were detected and expression of OXA-931 demonstrated a 2–64-fold increase in the minimum inhibitory concentration for β-lactam agents. Comparative genomic analysis revealed that most A. bereziniae strains did not carry any antimicrobial resistance genes (ARGs); however, some strains from China, Brazil, and India harbored six or more ARGs. Furthermore, A. bereziniae strains harbored conserved virulence genes. These results add valuable information regarding the spread of ARGs and mobile genetic elements that could be shared not only between A. bereziniae but also by other bacteria of clinical interest. This study also demonstrates that A. bereziniae can spill over from anthropogenic sources into natural environments and subsequently be transmitted to non-human hosts, making this a potential One Health bacteria that require close surveillance.

Disseminated fusariosis is treated with amphotericin B and voriconazole. To determine adequate therapy, the minimal inhibitory concentration (MIC) is used. However, MIC analysis is based on visual observation and requires a long period of fungal incubation. The measure of the minimal profile change concentration (MPCC) using MALDI-TOF MS is a quick spectral method that has presented good results in determining the antimicrobial resistance of yeasts. However, there is a lack of information on filamentous fungi. In the present work, 13 Fusarium spp. clinical isolates and two reference strains were used. MIC was obtained according to the M38-A2 protocol of the Clinical Laboratory Standards Institute, while MPPC was obtained following the initial steps of the M38-A2 protocol. Both Biotyper and the Rstudio environment were used to analyze mass spectra. For some fungal strains, the data obtained from the software MALDI Biotyper Compass 4.1 led to fuzzy heatmaps resulting in difficult interpretation, while heatmaps obtained using Rstudio tools generated better MPCC resolutions. Herein, 86.6% of the AMB MPCC values were highly correlated with the gold-standard AMB MIC. MALDI-TOF MS is a prominent tool used to determine MPCCs quicker, cost-effectively, and more accurately for Fusarium spp. strains. However, better statistical analyses could help measure the technique’s limit detection.

Next-Generation Sequencing (NGS) is becoming a reality in the clinical microbiology laboratory because it can speed diagnosis when compared to traditional culture based-methods and moreover, to aid in unravelling key virulence traits of important pathogens. Nonetheless, there are many limitations for its wide application in routine testing, as the requirement of high performance hardware and software to support bioinformatics analysis, as well as the expertise in different programming languages to perform the analyses. In this context, this review was drawn to synthesize some basic concepts involved in NGS for Whole-Genome Sequencing (WGS), based on two international straightforward efforts to standardize WGS data acquisition and processing in the clinical routine, the PulseNet International and the ENGAGE project, allied with other tools available for WGS analysis, beginning from the available sequencing platforms to the main user-friendly pipelines dedicated for the pathogen identification, including the use of properly databases to search for virulence factors, resistance genes and software resources for molecular typing of isolates.