Bacteriophage-Associated Antimicrobial Resistance Genes in Avian Pathogenic Escherichia coli Isolated from Brazilian Poultry.

Colibacillosis is a disease caused by Escherichia coli and remains a major concern in poultry production, as it leads to significant economic losses due to carcass condemnation and clinical symptoms. The development of antimicrobial resistance is a growing problem of worldwide concern. Lysogenic bacteriophages are effective vectors for acquiring and disseminating antibiotic resistance genes (ARGs). The aim of this study was to investigate the complete genome of Escherichia coli isolates from the femurs of Brazilian broiler chickens in order to investigate the presence of antimicrobial resistance genes associated with bacteriophages. Samples were collected between August and November 2021 from broiler batches from six Brazilian states. Through whole genome sequencing (WGS), data obtained were analyzed for the presence of antimicrobial resistance genes. Antimicrobial resistance genes against the aminoglycosides class were detected in 79.36% of the isolates; 74.6% had predicted sulfonamides resistance genes, 63.49% had predicted resistance genes against ß-lactams, and 49.2% of the isolates had at least one of the tetracycline resistance genes. Among the detected genes, 27 have been described in previous studies and associated with bacteriophages. The findings of this study highlight the role of bacteriophages in the dissemination of ARGs in the poultry industry.

Genomic Surveillance of SARS-CoV-2 in Healthcare Workers: A Critical Sentinel Group for Monitoring the SARS-CoV-2 Variant Shift.

SARS-CoV-2 genome surveillance is important for monitoring risk groups and health workers as well as data on new cases and mortality rate due to COVID-19. We characterized the circulation of SARS-CoV-2 variants from May 2021 to April 2022 in the state of Santa Catarina, southern Brazil, and evaluated the similarity between variants present in the population and healthcare workers (HCW). A total of 5291 sequenced genomes demonstrated the circulation of 55 strains and four variants of concern (Alpha, Delta, Gamma and Omicron-sublineages BA.1 and BA.2). The number of cases was relatively low in May 2021, but the number of deaths was higher with the Gamma variant. There was a significant increase in both numbers between December 2021 and February 2022, peaking in mid-January 2022, when the Omicron variant dominated. After May 2021, two distinct variant groups (Delta and Omicron) were observed, equally distributed among the five Santa Catarina mesoregions. Moreover, from November 2021 to February 2022, similar variant profiles between HCW and the general population were observed, and a quicker shift from Delta to Omicron in HCW than in the general population. This demonstrates the importance of HCW as a sentinel group for monitoring disease trends in the general population.

Trypanosoma evansi secretome carries potential biomarkers for Surra diagnosis.

Trypanosoma evansi is a parasite that is phylogenetically close to Trypanosoma brucei and is the causative agent of a disease known as surra. Surra is responsible for a high mortality rate in livestock and large economic losses in the Americas, Africa, and Asia. This work aimed to analyze in vitro secreted proteins from T. evansi and identify potential treatment and diagnostic biomarkers for surra diagnosis. Two groups were used. In one group the parasites were purified using a DEAE-Cellulose column and maintained in a secretion medium while in the other group the parasites were not purified. Each group was further divided to be maintained at either 37 °C or 27 °C. We identified 246 proteins through mass spectrometry and found that the temperature appears to modulate protein secretion. We found minimal variations in the protein pools from pure and non-purified sets. We observed an emphasis on proteins associated to vesicles, glycolysis, and cellular homeostasis through the enrichment of GO. Also, we found that most secretome proteins share homologous proteins with T. b. brucei, T. b. gambiense, T. vivax, T. equiperdum, and T. b. rhodesiense secretome but unique T. evansi epitopes with potential biomarkers for surra diagnosis were detected. SIGNIFICANCE: Trypanosoma evansi is a parasite of African origin that is phylogenetically close to Trypanosoma brucei. As with other trypanosomatids and blood parasites, its infection causes non-pathognomonic symptoms, which makes its diagnosis difficult. One great problem is the fact that no diagnostic test differentiates between Trypanosoma equiperdum and T. evansi, which is a problem in South America and Asia, and Africa. Thus, it is urgent to study the biochemistry of the parasite to discover proteins that can be used for differential diagnosis or be possible therapeutic targets. In addition, the study of the secretome can point out proteins that are used by the parasite in its interactions with the host, helping to understand the progression of the disease.

AnnotaPipeline: An integrated tool to annotate eukaryotic proteins using multi-omics data.

Assignment of gene function has been a crucial, laborious, and time-consuming step in genomics. Due to a variety of sequencing platforms that generates increasing amounts of data, manual annotation is no longer feasible. Thus, the need for an integrated, automated pipeline allowing the use of experimental data towards validation of in silico prediction of gene function is of utmost relevance. Here, we present a computational workflow named AnnotaPipeline that integrates distinct software and data types on a proteogenomic approach to annotate and validate predicted features in genomic sequences. Based on FASTA (i) nucleotide or (ii) protein sequences or (iii) structural annotation files (GFF3), users can input FASTQ RNA-seq data, MS/MS data from mzXML or similar formats, as the pipeline uses both transcriptomic and proteomic information to corroborate annotations and validate gene prediction, providing transcription and expression evidence for functional annotation. Reannotation of the available Arabidopsis thaliana, Caenorhabditis elegans, Candida albicans, Trypanosoma cruzi, and Trypanosoma rangeli genomes was performed using the AnnotaPipeline, resulting in a higher proportion of annotated proteins and a reduced proportion of hypothetical proteins when compared to the annotations publicly available for these organisms. AnnotaPipeline is a Unix-based pipeline developed using Python and is available at: https://github.com/bioinformatics-ufsc/AnnotaPipeline.

Different Swine Production Systems Can Shape Slurry Resistome at Mechanism and Class Levels Based on Swine Manure Evaluation.

Antimicrobial resistance is a major threat to public health. Antimicrobial use in animal husbandry is a major concern since it can favor an increase in antimicrobial resistance among farms. Herein, we aim to better understand and characterize the main resistome profiles in microbial communities found in pig farms. Sampling of swine manure was performed in two different timepoints (October 2019 and January 2020) in each of the 14 different swine farms, located in the mesoregion of Western Santa Catarina state in Brazil, a pole of swine product production of worldwide importance. Samples were divided into three groups: farms with the opened regimen and no usage of antimicrobials (F1; n = 10), farms with the closed regimen and usage of antimicrobials (F2; n = 16), and farms with the closed regimen and no usage of antimicrobials (F3; n = 2). The metagenomic evaluation was performed to obtain and identify genetic elements related to antimicrobial resistance using nanopore sequencing. We used ResistoXplorer software to perform composition, alpha and beta diversity, and clustering analysis. In addition, PCR reactions were performed to confirm the presence or absence of seven different beta-lactamase family genes and five phosphoethanolamine transferase gene variants clinically relevant. Our findings based on the identification of resistance genes at the mechanism level showed a prevalence of alteration of the drug target (72.3%) profile, followed by drug inactivation (17.5%) and drug efflux (10.1%). We identified predominantly aminoglycosides (45.3%), tetracyclines (15.9%), and multiclass (11,2%) resistance genes. PCoA analysis indicates differences between F1 and F2 profiles. F2 samples showed increased diversity when compared to the F1 group. In addition, herein we first report the identification of mcr-4 in a slurry sample (C1F1.1) in Santa Catarina State. In general, our findings reinforce that many factors on the practices of animal husbandry are involved in the resistome profile at the mechanism and class levels. Further studies to better understand microbiome and mobilome aspects of these elements are necessary to elucidate transmission pathways between different bacteria and environments.

Comparison of functional-oil blend and anticoccidial antibiotics effects on performance and microbiota of broiler chickens challenged by coccidiosis.

This study aimed to compare the effects of different levels of cashew nutshell liquid (CNSL) and castor oil (CNSL-castor oil) with growth-promoting antibiotics associated with anticoccidials in broiler chickens challenged with coccidiosis. In this work, 2520 one-day-old male broiler chicks (Cobb) were randomly assigned to 84 pens, containing 30 birds each. The experimental design was completely randomized, with seven treatments: enramycin (8 ppm), virginiamycin (16.5 ppm), and tylosin (55 ppm); different doses of CNSL-castor oil (0.5, 0.75, and 1.00 kg/t); and a control diet (without additives). All treatments received semduramicin + nicarbazin (500 g/t; Aviax® Plus) from 0 to 28 d and monensin sodium (100 ppm; Elanco) from 29 to 35 days of age, when the feed was without antibiotics. The challenge was introduced at 14 days of age by inoculating broiler chickens with sporulated Eimeria tenella, Eimeria acervulina, and Eimeria maxima oocysts via oral gavage. In addition to performance parameters, intestinal contents were collected at 28 and 42 days of age for microbiota analysis by sequencing the 16s rRNA in V3 and V4 regions using the Illumina MiSeq platform. Taxonomy was assigned using the SILVA database (v. 138) with QIIME2 software (v. 2020.11). After one week of challenge, the broilers that received tylosin had a higher body weight gain (BWG) than those in the control group (p < 0.05), while the other treatments presented intermediate values. At 28 d, the BWG was lower for the control, CNSL-Castor oil 0.5 kg/t, enramycin, and virginiamycin treatments than that in the tylosin treatment. The inclusion of CNSL-Castor oil at concentrations of 0.75 and 1 kg/t acted as an intermediate treatment (p < 0.05). For alpha diversity, using the Shannon index, it was possible to observe the effect of age, with substantial diversity at 42 d. The Firmicutes phylum had the highest abundance, with values between 84.33% and 95.16% at 42 d. Tylosin showed better performance indices than other treatments. CNSL-castor oil treatments with concentrations of 0.75 and 1 kg/t showed similar results to those of enramycin and virginiamycin. Furthermore, CNSL-castor oil acted as a modulator of intestinal microbiota, reducing the abundance of pathogenic bacteria.

EpiBuilder: A Tool for Assembling, Searching, and Classifying B-Cell Epitopes.

Epitopes are portions of a protein that are recognized by antibodies. These small amino acid sequences represent a significant breakthrough in a branch of bioinformatics called immunoinformatics. Various software are available for linear B-cell epitope (BCE) prediction such as ABCPred, SVMTrip, EpiDope, and EpitopeVec; a well-known BCE predictor is BepiPred-2.0. However, despite the prediction, there are several essential steps, such as epitope assembly, evaluation, and searching for epitopes in other proteomes. Here, we present EpiBuilder (https://epibuilder.sourceforge.io), a user friendly software that assists in epitope assembly, classifying and searching using input results of BepiPred-2.0. EpiBuilder generates several output results from these data and supports a proteome-wide processing approach. In addition, this software provides the following features: Chou & Fasman beta-turn prediction, Emini surface accessibility prediction, Karplus and Schulz flexibility prediction, Kolaskar and Tongaonkar antigenicity, Parker hydrophilicity prediction, N-glycosylation domains, and hydropathy. These information generate a unique topology for each epitope, visually demonstrating its characteristics. The software can search the entire epitope sequence in various FASTA files, and it allows to use BLASTP to identify epitopes that eventually have sequence variations. As an EpiBuilder application, we developed a epitope dataset from the protozoan Trypanosoma brucei gambiense, the gram-positive bacterium Clostridioides difficile, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Exoproteomic analysis of two MLST clade 2 strains of Clostridioides difficile from Latin America reveal close similarities.

Clostridioides difficile BI/NAP1/ribotype 027 is an epidemic hypervirulent strain found worldwide, including in Latin America. We examined the genomes and exoproteomes of two multilocus sequence type (MLST) clade 2 C. difficile strains considered hypervirulent: ICC-45 (ribotype SLO231/UK[CE]821), isolated in Brazil, and NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica. C. difficile isolates were cultured and extracellular proteins were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Genomic analysis revealed that these isolates shared most of the gene composition. Only 83 and 290 NAP1/027 genes were considered singletons in ICC-45 and NAP1/027, respectively. Exoproteome analysis revealed 197 proteins, of which 192 were similar in both strains. Only five proteins were exclusive to the ICC-45 strain. These proteins were involved with catalytic and binding functions and indirectly interacted with proteins related to pathogenicity. Most proteins, including TcdA, TcdB, flagellin subunit, and cell surface protein, were overrepresented in the ICC-45 strain; 14 proteins, including mature S-layer protein, were present in higher proportions in LIBA5756. Data are available via ProteomeXchange with identifier PXD026218. These data show close similarity between the genome and proteins in the supernatant of two strains with hypervirulent features isolated in Latin America and underscore the importance of epidemiological surveillance of the transmission and emergence of new strains.