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Klebsiella aerogenes is an important opportunistic pathogen with the potential to develop resistance against last-line antibiotics, such as carbapenems, limiting the treatment options. Here, we investigated the antibiotic resistance profiles of 10 K. aerogenes strains isolated from patient samples in the intensive-care unit of a Brazilian tertiary hospital using conventional PCR and a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the bla KPC-2 and bla NDM-1 genes simultaneously. All isolates were completely resistant to β-lactam antibiotics, including ertapenem, imipenem, and meropenem with differencing levels of resistance to aminoglycosides, quinolones, and tigecycline also observed. Half of the strains studied were classified as multidrug-resistant. The carbapenemase-producing isolates carried many genes of interest including: β-lactams ( bla NDM-1 , bla KPC-2 , bla TEM-1 , bla CTX-M-1 group, bla OXA-1 group and bla SHVvariants in 20-80% of the strains), aminoglycoside resistance genes [ aac(6')-Ib and aph ( 3')-VI , 70 and 80%], a fluoroquinolone resistance gene ( qnrS , 80%), a sulfonamide resistance gene ( sul-2 , 80%) and a multidrug efflux system transporter ( mdtK , 70%) while all strains carried the efflux pumps Acr (subunit A) and tolC . Moreover, we performed a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the bla KPC-2 and bla NDM-1 genes simultaneously. The draft genome assembly of the CRK317 had a total length of 5,462,831 bp and a GC content of 54.8%. The chromosome was found to contain many essential genes. In silico analysis identified many genes associated with resistance phenotypes, including β-lactamases ( bla OXA-9 , bla TEM-1 , bla NDM-1 , bla CTX-M-15 , bla AmpC-1 , bla AmpC-2 ), the bleomycin resistance gene ( ble MBL ), an erythromycin resistance methylase ( ermC ), aminoglycoside-modifying enzymes [ aac(6') - Ib , aadA/ant(3") - Ia , aph(3')-VI ], a sulfonamide resistance enzyme ( sul-2 ), a chloramphenicol acetyltransferase ( catA- like), a plasmid-mediated quinolone resistance protein ( qnrS1 ), a glutathione transferase ( fosA ), PEtN transferases ( eptA , eptB ) and a glycosyltransferase ( arnT ). We also detected 22 genomic islands, eight families of insertion sequences, two putative integrative and conjugative elements with a type IV secretion system, and eight prophage regions. This suggests the significant involvement of these genetic structures in the dissemination of antibiotic resistance. The results of our study show that the emergence of carbapenemase-producing K. aerogenes , co-harboring bla KPC-2 and bla NDM-1 , is a worrying phenomenon which highlights the importance of developing strategies to detect, prevent, and control the spread of these microorganisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rodrigues, Nunes, Soares, Ferreira, Damas, Laprega, Shilling, Campos, Costa, Malavazi, Cunha and Pranchevicius.)

The red blood cells (RBCs) are essential to transport oxygen (O 2 ) and nutrients throughout the human body. Changes in the structure or functioning of the erythrocytes can lead to several deficiencies, such as hemolytic anemias, in which an increase in reactive oxidative species generation is involved in the pathophysiological process, playing a significant role in the severity of several clinical manifestations. There are important lines of defense against the damage caused by oxidizing molecules. Among the antioxidant molecules, the enzyme peroxiredoxin (Prx) has the higher decomposition power of hydrogen peroxide, especially in RBCs, standing out because of its abundance. This review aimed to present the recent findings that broke some paradigms regarding the three isoforms of Prxs found in RBC (Prx1, Prx2, and Prx6), showing that in addition to their antioxidant activity, these enzymes may have supplementary roles in transducing peroxide signals, as molecular chaperones, protecting from membrane damage, and maintenance of iron homeostasis, thus contributing to the overall survival of human RBCs, roles that seen to be disrupted in hemolytic anemia conditions., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Leishmaniasis continues to pose a substantial health burden in 97 countries worldwide. The progression and outcome of Leishmania infection are influenced by various factors, including the cytokine milieu, the skin microbiota at the infection site, the specific Leishmania species involved, the genetic background of the host, and the parasite load. In endemic regions to leishmaniasis, only a fraction of individuals infected actually develops the disease. Overexpression of IL-13 in naturally resistant C57BL/6 mice renders them susceptible to L. major infection. Haplotypes constructed from several single nucleotide variant (SNV) along a chromosome fragment may provide insight into any SNV near the fragment that may be genuinely associated with a phenotype in genetic association studies., Methods: We investigated nine SNVs (SNV1rs1881457A>C, SNV2rs1295687C>G, SNV3rs2069744C>T, SNV4rs2069747C>T, SNV5rs20541A>G, SNV6rs1295685A>G, SNV7rs848A>C, SNV8rs2069750G >C, and SNV9rs847T>C) spanning the entire IL13 gene in patients with L. guyanensis cutaneous leishmaniasis ( Lg -CL)., Results: Our analysis did not reveal any significant association between the SNVs and susceptibility/protection against Lg -CL development. However, haplotype analysis, excluding SNV4rs2069747 and SNV8rs2069750 due to low minor allele frequency, revealed that carriers of the haplotype CCCTAAC had a 93% reduced likelihood developing Lg-CL. Similarly, the haplotypes ACCCGCT (ORadj=0.02 [95% CI 0.00-0.07]; p -value, 6.0×10 -19 ) and AGCTAAC (ORadj=0.00[95% CI 0.00-0.00]; p -value 2.7×10 -12 ) appeared to provide protection against the development of Lg -CL. Conversely, carriers of haplotype ACCTGCC have 190% increased likelihood of developing Lg -CL (ORadj=2.9 [95%CI 1.68-5.2]; p -value, 2.5×10 -6 ). Similarly, haplotype ACCCAAT (ORadj=2.7 [95%CI 1.5-4.7]; p -value, 3.2×10 -5 ) and haplotype AGCCGCC are associated with susceptibility to the development of Lg -CL (ORadj=1.7[95%CI 1.04-2.8]; p -value, 0.01). In our investigation, we also found a correlation between the genotypes of rs2069744, rs20541, rs1295685, rs847, and rs848 and plasma IL-5 levels among Lg -Cl patients. Furthermore, rs20541 showed a correlation with plasma IL-13 levels among Lg -Cl patients, while rs2069744 and rs848 showed a correlation with plasma IL-4 levels among the same group., Conclusions: Overall, our study identifies three haplotypes of IL13 associated with resistance to disease development and three haplotypes linked to susceptibility. These findings suggest the possibility of a variant outside the gene region that may contribute, in conjunction with other genes, to differences in susceptibility and partially to the pathology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Junior, Souza, Silva, Silva, Nascimento, Souza, Cunha, Batista, Neto, Guerra and Ramasawmy.)

Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 ( xylA1 ) and XAC4225 ( xylA2 ), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes ( xylA1 and/or xylA2 ) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence.

Background: Swine production expanded in the last decades. Efforts have been made to improve meat production and to understand its relationship to pig gut microbiota. Copper (Cu) is a usual supplement to growth performance in animal production. Here, two performance studies were conducted to investigate the effects of three different sources of Cu on the microbiota of piglets. A total of 256 weaned piglets were randomly allocated into 4 treatments (10 replicates per treatment of 4 piglets per pen in Trial 1 and 8 replicates of 3 piglets per pen in Trial 2). Treatments included a control group (fed 10 mg/kg of Cu from CuSO 4 ), a group fed at 160 mg/kg of Copper (II) sulfate (CuSO 4 ) or tri-basic copper chloride (TBCC), and a group fed with Cu methionine hydroxy analogue chelated (Cu-MHAC) at 150, 80, and 50 mg/kg in Phases 1 (24-35 d), 2 (36-49 d), and 3 (50-70 d), respectively. At 70 d, the cecum luminal contents from one pig per pen were collected and polled for 16 S rRNA sequencing (V3/V4 regions). Parameters were analyzed in a completely randomized block design, in which each experiment was considered as a block., Results: A total of 1337 Operational Taxonomic Units (OTUs) were identified. Dominance and Simpson ecological metrics were statistically different between control and treated groups (P < 0.10) showing that different Cu sources altered the gut microbiota composition with the proliferation of some bacteria that improve gut health. A high abundance of Prevotella was observed in all treatments while other genera were enriched and differentially modulated, according to the Cu source and dosage. The supplementation with Cu-MHAC can modify a group of bacteria involved in feed efficiency (FE) and short chain fatty acids (SCFA) production (Clostridium XIVa, Desulfovibrio, and Megasphera). These bacteria are also important players in the activation of ghrelin and growth hormones that were previously reported to correlate with Cu-MHAC supplementation., Conclusions: These results indicated that some genera seem to be directly affected by the Cu source offered to the animals. TBCC and Cu-MHAC (even in low doses) can promote healthy modifications in the gut bacterial composition, being a promising source of supplementation for piglets., (© 2023. The Author(s).)

This study aimed to establish the importance of ergothioneine (ERT) in the erythroid adaptation mechanisms by appraising the expression levels of redox-related genes associated with the PI3K/AKT/FoxO3 and Nrf2-ARE pathways using K562 cells induced to erythroid differentiation and H 2 O 2 -oxidative stress. Cell viability and gene expression were evaluated. Two concentrations of ERT were assessed, 1 nM (C1) and 100 µM (C2), with and without stress induction (100 µM H 2 O 2 ). Assessments were made in three periods of the cellular differentiation process (D0, D2, and D4). The C1 treatment promoted the induction of FOXO3 (D0 and 2), PSMB5 , and 6 expressions (D4); C1 + H 2 O 2 treatment showed the highest levels of NRF2 transcripts, KEAP1 (D0), YWHAQ (D2 and 4), PSMB5 (D2) and PSMB6 (D4); and C2 + H 2 O 2 (D2) an increase in FOXO3 and MST1 expression, with a decrease of YWHAQ and NRF2 was observed. in C2 + H 2 O 2 (D2) an increase in FOXO3 and MST1 , with a decrease in YWHAQ and NRF2 was observed All ERT treatments increased gamma-globin expression. Statistical multivariate analyzes highlighted that the Nrf2-ARE pathway presented a greater contribution in the production of PRDX1 , SOD1 , CAT , and PSBM5 mRNAs, whereas the PI3K/AKT/FoxO3 pathway was associated with the PRDX2 and TRX transcripts. In conclusion, ERT presented a cytoprotective action through Nrf2 and FoxO3, with the latter seeming to contribute to erythroid proliferation/differentiation.

Melatonin (MEL) presents well-documented pleiotropic actions against oxidative stress (OS), acting indirectly through activation of transcription factors, e.g., FoxO3 and Nrf2. Thus, this study aimed to investigate the possible modulating effects of MEL on the redox signaling pathways PI3K/AKT/FoxO3 and Keap1/Nrf2/ARE in K562 erythroleukemic cells subjected to OS induction. For this, the viability, and transcript levels of genes involved in redox adaptation were evaluated in K562 cells in different periods of erythroid differentiation: under OS induction by hydrogen peroxide (100 µM H 2 O 2 ); treated with 1 nM (C1) and 1 mM (C2) MEL; and associated or not with stress induction. We observed a restoration of physiological levels of Nrf2 in both MEL concentrations under OS. The C1 was related to enhanced expression of antioxidant and proteasome genes through the Nrf2-ARE pathway, while C2 to the induction of FOXO3 expression, suggesting an involvement with apoptotic pathway, according to BIM transcript levels. The effects of MEL administration in these cells showed a period and dose-dependent pattern against induced-OS, with direct and indirect actions through different pathways of cellular adaptation, reinforcing the importance of this indolamine in the regulation of cellular homeostasis, being a promising therapeutic alternative for diseases that present an exacerbated OS.

Staphylococcus aureus mastitis constitutes a serious threat to dairy cows. The reasons why available vaccines are not fully effective remain poorly understood; thus, in the present study, we investigated CD4 + and CD8 + T lymphocyte proliferation in dairy cows vaccinated with a polyvalent mastitis vaccine that had distinct precedent Staphylococcus aureus mastitis. We studied 17 S. aureus-infected dairy cows (11 vaccinated and six unvaccinated) and eight vaccinated healthy dairy cows with no previous S. aureus mastitis infections. Flow cytometry was used to assess lymphocyte proliferation using an anti-Ki67 antibody, and monoclonal antibodies were used to identify T cell subsets. S. aureus-infected cows exhibited reduced overall lymphocyte proliferation, including CD4 + T lymphocyte proliferation, and memory lymphocyte proliferation in response to S. aureus isolate stimulus. Immunization did not influence the expansion of blood lymphocyte populations. Furthermore, CD8 + T cells, memory CD8 + T lymphocytes, and effector memory CD8 + T lymphocytes displayed reduced proliferation 21 days after the third vaccine dose compared with before vaccination at time zero. The present data demonstrates an overall negative regulation of the T-cell response suggesting its detrimental impact leading to the persistence of S. aureus intramammary infections. Furthermore, the lack of vaccination effect on T-cell mediated immunity (e.g., proliferation) may be related to poor vaccine efficacy., Competing Interests: Declaration of interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background & Aims: Several studies have shown conflicting results for the relationship between vitamin D deficiency and COVID-19 outcomes. Here, we aimed to evaluate whether plasma 25(OH)D levels predict mortality in adults admitted with COVID-19, considering potential confounders., Methods: We conducted a retrospective cohort study that included 115 adults (age 62.1 ± 17.6 years, 65 males) admitted to a Brazilian public hospital for severely symptomatic COVID-19. Subjects were classified into two groups according to their plasma levels of 25(OH)D: sufficiency (≥50 nmol/L) and the deficiency (<50 nmol/L). The diagnosis of COVID-19 was performed using real-time polymerase chain reaction (qPCR). In addition, direct competitive chemiluminescence immunoassay assessed serum 25(OH)D levels., Results: The all-cause 30-day mortality was 13.8% (95% CI: 6.5%-21%) in the group of patients with sufficient plasma 25(OH)D levels and 32.1% (95% CI: 14.8%-49.4%) among those with deficient plasma 25(OH)D levels. Cox regression showed that plasma 25(OH)D levels remained a significant predictor of mortality even after adjusting for the covariates sex, age, length of the delay between symptom onset and hospitalization, and disease severity (HR = 0.98, 95% CI: 0.96-1.00; p = 0.02)., Conclusion: Vitamin D deficiency predicts higher mortality risk in adults with COVID-19., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including β-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to β-lactamases ( bla IND- 13 , bla CIA- 3 , bla TEM- 116 , bla OXA- 209 , bla VEB- 15 ), quinolone ( mcb G), tigecycline ( tet (X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides ( Ran A/ Ran B), and colistin ( Hly D/ Tol C). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes , which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Damas, Ferreira, Campanini, Soares, Campos, Laprega, Soares da Costa, Freire, Pitondo-Silva, Cerdeira, Cunha and Pranchevicius.)

Objective: To compare blind and endoscopic-guided techniques for orotracheal intubation in rabbits and the number of intubation attempts with laryngeal/tracheal damage., Study Design: Prospective, randomized experimental study., Animals: A total of 24 healthy, intact female New Zealand White rabbits, weighing 2.2 ± 0.2 kg (mean ± standard deviation)., Methods: Rabbits were randomly assigned to blind (group B) or endoscopic-guided (group E) orotracheal intubation with a 2.0 mm internal diameter uncuffed tube. Intramuscular (IM) alfaxalone (7 mg kg -1 ), hydromorphone (0.1 mg kg -1 ) and dexmedetomidine (0.005 mg kg -1 ) were administered, and additional IM alfaxalone (3-5 mg kg -1 ) and dexmedetomidine (0.025 mg kg -1 ) were administered to rabbits with strong jaw tone. An intubation attempt was defined as the advancement of the endotracheal tube from the incisors to the laryngeal entrance. Tracheal intubation was confirmed via capnography and anesthesia was maintained with isoflurane for 2 hours. Following euthanasia, laryngeal and tracheal tissues were submitted for histopathology. Quality of anesthesia for orotracheal intubation, intubation procedure and tissue damage were numerically scored. Data were analyzed using Poisson regression, Spearman's correlation, t test, mixed anova, Mann-Whitney U test, Friedman and Chi square tests as appropriate., Results: Median (range) intubation attempts were 2 (1-8) and 1 (1-3) for groups B and E, respectively. More rabbits in group E (91.6%) required additional alfaxalone and dexmedetomidine than in group B (16.7%). Median (range) cumulative histopathology scores were 6 (3-10) and 6 (2-9) for groups B and E, respectively. Scores were highest in the cranial trachea, but there was no difference between groups and no correlation between laryngeal/tracheal damage and the number of intubation attempts., Conclusions and Clinical Relevance: Both orotracheal intubation techniques were associated with laryngeal/tracheal damage. Although blind orotracheal intubation was associated with a higher number of attempts, the tissue damage was similar between groups., (Copyright © 2022 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved.)

The fermented beverage industry is always pursuing alternatives to make products that delight consumers with special or unique characteristics. The identification and improvement of new yeast strains emerge as an opportunity; however, wild strains usually have a limitation in maltose fermentation and/or off-flavors production. Here we report the production of a Blond-style ale beer using a bioethanol isolated strain (LBGA-287) with flavor complexity approved in sensorial panels. LBGA-287 also showed an increase in maltose consumption, growth and fermentation rates when compared to the commercial yeast. Using qPCR analysis, genes related to the (i) efficiency of fermentation (ii) production of aromas/off-flavors, and (iii) metabolization of carbohydrates were found as differentially expressed in the isolated strains when compared to industrial yeast. This suggests that LBGA-287 could have an important impact on beer production, improving brewing efficiency, quality and diversity of this beverage, and most importantly satisfying the final consumer., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Serratia marcescens is now an important opportunistic pathogen that can cause serious infections in hospitalized or immunocompromised patients. Here, we used extensive bioinformatic analyses based on reverse vaccinology and subtractive proteomics-based approach to predict potential vaccine candidates against S. marcescens . We analyzed the complete proteome sequence of 49 isolate of Serratia marcescens and identified 5 that were conserved proteins, non-homologous from human and gut flora, extracellular or exported to the outer membrane, and antigenic. The identified proteins were used to select 5 CTL, 12 HTL, and 12 BCL epitopes antigenic, non-allergenic, conserved, hydrophilic, and non-toxic. In addition, HTL epitopes were able to induce interferon-gamma immune response. The selected peptides were used to design 4 multi-epitope vaccines constructs (SMV1, SMV2, SMV3 and SMV4) with immune-modulating adjuvants, PADRE sequence, and linkers. Peptide cleavage analysis showed that antigen vaccines are processed and presented via of MHC class molecule. Several physiochemical and immunological analyses revealed that all multiepitope vaccines were non-allergenic, stable, hydrophilic, and soluble and induced the immunity with high antigenicity. The secondary structure analysis revealed the designed vaccines contain mainly coil structure and alpha helix structures. 3D analyses showed high-quality structure. Molecular docking analyses revealed SMV4 as the best vaccine construct among the four constructed vaccines, demonstrating high affinity with the immune receptor. Molecular dynamics simulation confirmed the low deformability and stability of the vaccine candidate. Discontinuous epitope residues analyses of SMV4 revealed that they are flexible and can interact with antibodies. In silico immune simulation indicated that the designed SMV4 vaccine triggers an effective immune response. In silico codon optimization and cloning in expression vector indicate that SMV4 vaccine can be efficiently expressed in E. coli system. Overall, we showed that SMV4 multi-epitope vaccine successfully elicited antigen-specific humoral and cellular immune responses and may be a potential vaccine candidate against S. marcescens . Further experimental validations could confirm its exact efficacy, the safety and immunogenicity profile. Our findings bring a valuable addition to the development of new strategies to prevent and control the spread of multidrug-resistant Gram-negative bacteria with high clinical relevance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Damas, Mazur, Freire, Cunha and Pranchevicius.)

Local anesthetics provide analgesia and can be incorporated into multimodal anesthetic protocols. They work by blocking the voltage-dependent sodium ion channels along neurons that mediate nociception. Systemically, these drugs can be cardiotoxic in a dose-dependent manner. Lidocaine and bupivacaine are the most commonly used local anesthetics and their use has been reported in all classes of vertebrates. Despite anecdotal reports to the contrary, zoologic companion animals are unlikely to be more susceptible to the cardiotoxic effects of local anesthetics than domestic small animals. Local anesthetics can be clinically useful for analgesia and anesthesia in zoologic companion animal practice., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: To determine the frequency, severity and duration of adverse events including myoclonus, pain on injection, hypersalivation, regurgitation and apnoea after administration of midazolam or saline followed by etomidate in hydromorphone premedicated dogs., Materials and Methods: Dogs undergoing elective dental prophylaxis or soft tissue surgeries were enrolled in this randomised trial. Dogs were premedicated with hydromorphone 0.1 mg/kg IV. Sixty seconds later, midazolam 0.3 mg/kg or saline at an equivalent volume was administered IV. Sixty seconds after that, etomidate 1.5 mg/kg IV was administered over 60 seconds. Additional doses of 0.5 mg/kg etomidate were administered until endotracheal intubation was successful. Observers were blinded to the treatment. Frequency, duration and a severity score of 0 to 3 were recorded for myoclonus, pain on injection, hypersalivation and regurgitation. Duration of apnoea and frequency of any additional complications was recorded., Results: Forty variable breed healthy dogs were enrolled in the study. Myoclonus, pain on injection, regurgitation, hypersalivation, gagging, tachypnoea and pigmenturia occurred, respectively, in 10%, 40%, 0%, 15%, 35%, 25% and 5% of dogs in the saline group and 0%, 65%, 0%, 10%, 45%, 15% and 5% of dogs in the midazolam group. Apnoea occurred for 115 seconds (range 0 to 660 seconds) and 160 seconds (range 0 to 600 seconds) in the saline and midazolam groups, respectively. Two dogs developed pigmenturia. The trial was stopped early due to the occurrence of pigmenturia., Clinical Significance: Due to early stopping of the trial, the predefined sample size was not reached. Further investigation is needed to determine if midazolam reduced the incidence of adverse events or improved the induction quality when combined with hydromorphone and etomidate., (© 2021 British Small Animal Veterinary Association.)

Aspergillus fumigatus produces diverse secondary metabolites whose biological functions and regulation remain to be understood. Despite the importance of the conidia for this fungus, the role of the conidia-born metabolite fumiquinazoline C (FqC) is unclear. Here, we describe a dual function of the cell-wall integrity pathway in regulating FqC biosynthesis dictated by the MAPK kinase MpkA, which phosphorylates one of the nonribosomal peptide synthetases enzymes of the cluster (FmqC), and the transcription factor RlmA, which directly regulates the expression of fmq genes. Another level of crosstalk between the FqC regulation and the cell physiology is described since the deletion of the stress-responsive transcription factor sebA provokes derepression of the fmq cluster and overproduction of FqC. Thus, we describe a mechanism by which A. fumigatus controls FqC biosynthesis orchestrated by MpkA-RlmA and SebA and hence enabling survival and adaptation to the environmental niche, given that FqC is a deterrent of ameba predation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The deleterious effects of human-induced climate change have long been predicted. However, the imminent emergence and spread of new diseases, including fungal infections through the rise of thermotolerant strains, is still neglected, despite being a potential consequence of global warming. Thermotolerance is a remarkable virulence attribute of the mold Aspergillus fumigatus . Under high-temperature stress, opportunistic fungal pathogens deploy an adaptive mechanism known as heat shock (HS) response controlled by heat shock transcription factors (HSFs). In eukaryotes, HSFs regulate the expression of several heat shock proteins (HSPs), such as the chaperone Hsp90, which is part of the cellular program for heat adaptation and a direct target of HSFs. We recently observed that the perturbation in cell wall integrity (CWI) causes concomitant susceptibility to elevated temperatures in A. fumigatus , although the mechanisms underpinning the HS response and CWI cross talking are not elucidated. Here, we aim at further deciphering the interplay between HS and CWI. Our results show that cell wall ultrastructure is severely modified when A. fumigatus is exposed to HS. We identify the transcription factor HsfA as essential for A. fumigatus viability, thermotolerance, and CWI. Indeed, HS and cell wall stress trigger the coordinated expression of both hsfA and hsp90 . Furthermore, the CWI signaling pathway components PkcA and MpkA were shown to be important for HsfA and Hsp90 expression in the A. fumigatus biofilms. Lastly, RNA-sequencing confirmed that hsfA regulates the expression of genes related to the HS response, cell wall biosynthesis and remodeling, and lipid homeostasis. Our studies collectively demonstrate the connection between the HS and the CWI pathway, with HsfA playing a crucial role in this cross-pathway regulation, reinforcing the importance of the cell wall in A. fumigatus thermophily., Competing Interests: MD is a Co-Founder and Chief Scientific Officer (CSO) of MicroRid Technologies Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fabri, Rocha, Fernandes, Persinoti, Ries, Cunha, Goldman, Del Poeta and Malavazi.)

Termites are well recognized by their complex development trajectories, involving dynamic differentiation process between non-reproductive castes, workers and soldiers. These insects are associated with endosymbiotic microorganisms, which help in lignocellulose digestion and nitrogen metabolism. Aiming to identify genes harbouring biotechnological potential, we analyzed workers and soldiers RNA-Seq data of three neotropical termites: Heterotermes tenuis (Isoptera: Rhinotermitidae), Velocitermes heteropterus (Isoptera: Termitidae) and Cornitermes cumulans (Isoptera: Termitidae). We observed differences in the microbiota associated with each termite family, and found protists' genes in both Termitidae species. We found an opposite pattern of caste-biased gene expression between H. tenuis and the termitids studied. Moreover, the two termitids are considerably different concerning the number of differentially expressed genes (DEGs). Functional annotation indicated considerable differences in caste-biased gene content between V. heteropterus and C. cumulans, even though they share similar diet and biological niche. Among the most DEGs, we highlighted those involved in caste differentiation and cellulose digestion, which are attractive targets for studying more efficient technologies for termite control, biomass digestion and other biotechnological applications., (© 2020 The Royal Entomological Society.)

The initiation of Aspergillus fumigatus infection occurs via dormant conidia deposition into the airways. Therefore, conidial germination and subsequent hyphal extension and growth occur in a sustained heat shock (HS) environment promoted by the host. The cell wall integrity pathway (CWIP) and the essential eukaryotic chaperone Hsp90 are critical for fungi to survive HS. Although A. fumigatus is a thermophilic fungus, the mechanisms underpinning the HS response are not thoroughly described and important to define its role in pathogenesis, virulence and antifungal drug responses. Here, we investigate the contribution of the CWIP in A. fumigatus thermotolerance. We observed that the CWIP components PkcA, MpkA and RlmA are Hsp90 clients and that a PkcA G579R mutation abolishes this interaction. PkcA G579R also abolishes MpkA activation in the short-term response to HS. Biochemical and biophysical analyses indicated that Hsp90 is a dimeric functional ATPase, which has a higher affinity for ADP than ATP and prevents MpkA aggregation in vitro. Our data suggest that the CWIP is constitutively required for A. fumigatus to cope with the temperature increase found in the mammalian lung environment, emphasising the importance of this pathway in supporting thermotolerance and cell wall integrity., (© 2020 John Wiley & Sons Ltd.)

Objectives: In this randomized open-label trial pilot study we assessed the antiviral effects and safety of various doses of ivermectin in patients with mild clinical symptoms of COVID-19., Methods: Patients were randomly assigned to receive standard of care (SOC) treatment at hospital admission; SOC plus ivermectin 100 mcg/kg; SOC plus ivermectin 200 mcg/kg; or SOC plus ivermectin 400 mcg/kg. The primary assessed endpoint was the proportion of patients who achieved two consecutive negative SARS-CoV-2 RT PCR tests within 7 days of the start of the dosing period. This study was registered at ClinicalTrials.gov (NCT04431466)., Results: A total of 32 patients were enrolled and randomized to treatment. SOC treatment together with ivermectin did not result in any serious adverse events. All patients exhibited a reduction in SARS-CoV-2 viral load within 7 days; however, those who received ivermectin had a more consistent decrease as compared to the SOC alone group, characterized by a shorter time for obtaining two consecutive negative SARS-CoV-2 RT PCR tests., Conclusions: Ivermectin is safe in patients with SARS-CoV-2, reducing symptomatology and the SARS-CoV-2 viral load. This antiviral effect appears to depend on the dose used, and if confirmed in future studies, it suggests that ivermectin may be a useful adjuvant to the SOC treatment in patients with mild COVID-19 symptoms., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Background: Alzheimer's disease (AD) is the main cause of dementia and it is a progressive neurogenerative disease characterized by the accumulation of neurofibrillary tangles and senile plaques. There is currently no cure; however, some treatments are available to slow down the progression of the disease, including gene therapy, which has been investigated to have great potential for the treatment of AD., Objective: The aim of this review was to identify the efficacy of gene therapy to restore cognition in AD., Methods: A systematic review was carried out using papers published up to May 2020 and available in the Web of Science, Scopus, and Medline/PUBMED databases. Articles were considered for inclusion if they were original researches that investigated the effects of gene therapy on cognition in AD. The methodological quality of the selected studies was evaluated using the Risk of Bias Tool for Animal Intervention Studies (SYRCLE's Rob tool) and the Jadad Scale., Results: Most preclinical studies obtained positive results in improving memory and learning in mice that underwent treatment with gene therapy. On the other hand, clinical studies have obtained inconclusive results related to the delivery methods of the viral vector used in gene therapy., Conclusion: Gene therapy has shown a great potential for the treatment of AD in preclinical trials, but results should be interpreted with caution since preclinical studies presented limitations to predict the efficacy of the treatment outcome in humans., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In the present study, we aimed to determine the antimicrobial resistance and molecular typing of Staphylococcus aureus recovered from transient and persistent intramammary infections and nares/muzzles in dairy cows. We investigated the antimicrobial resistance of 189 S. aureus strains using a broad antimicrobial susceptibility profile. Furthermore, 107 S. aureus isolates were strain-typed using staphylococcal protein-A (spa) typing. A large proportion of strains exhibited multidrug resistance to antimicrobials, including resistance to critically important antimicrobials, although no methicillin-resistant S. aureus strains were found. Our study did not strengthen the idea that extramammary niches (i.e., nares/muzzles) are an important source of S. aureus for bovine mastitis. A discrepancy in the antimicrobial resistance between S. aureus strains isolated from nares/muzzles and milk samples was observed. Furthermore, S. aureus isolates from transient and persistent intramammary infections (IMIs) did not differ by spa typing, suggesting that the persistence of bovine IMIs was determined by cow factors. Thus, the high level of multidrug-resistant S. aureus found in the two herds, considered together with the predominance of a well udder-adapted S. aureus strain, may contribute to our knowledge of the history of the high prevalence of mastitis caused by S. aureus , which is of great concern for animal and public health.

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. A. fumigatus was isolated from cultures and identified in high incidence from patient sputum samples. Candida albicans, Penicillium sp., Hanseniaspora sp., Torulaspora delbrueckii , and Talaromyces amestolkiae were isolated sporadically. Metataxonomics and metagenomics detected fungal reads ( Saccharomyces cerevisiae , A. fumigatus , and  Schizophyllum sp.) in one sputum sample. The main pathogenic bacteria identified were Staphylococcus aureus , Pseudomonas aeruginosa , Burkholderia cepacia complex, and Achromobacter xylosoxidans . The canonical core CF microbiome is composed of species from the genera Streptococcus, Neisseria, Rothia, Prevotella , and Haemophilus . 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., (Copyright © 2020 de Almeida, Capizzani, Tonani, Grizante Barião, da Cunha, De Martinis, Torres and von Zeska Kress.)

Objective: To investigate the effects of a priming dose of alfaxalone on the total anesthetic induction dose for and cardiorespiratory function of sedated healthy cats., Animals: 8 healthy adult cats., Procedures: For this crossover study, cats were sedated with dexmedetomidine and methadone administered IM. Cats next received a priming induction dose of alfaxalone (0.25 mg/kg, IV) or saline (0.9% NaCl) solution (0.025 mL/kg, IV) over 60 seconds and then an induction dose of alfaxalone (0.5 mg/kg/min, IV) until orotracheal intubation was achieved. Cardiorespiratory variables were recorded at baseline (immediately prior to priming agent administration), immediately after priming agent administration, after orotracheal intubation, and every 2 minutes until extubation. The total induction dose of alfaxalone was compared between the 2 priming agents., Results: Mean ± SD total anesthetic induction dose of alfaxalone was significantly lower when cats received a priming dose of alfaxalone (0.98 ± 0.28 mg/kg), compared with when cats received a priming dose of saline solution (1.41 ± 0.17 mg/kg). Mean arterial blood pressure was significantly higher when alfaxalone was used as the priming dose. No cats became apneic or had a hemoglobin oxygen saturation of < 90%. Expired volume per minute was not significantly different between the 2 priming agents., Conclusions and Clinical Relevance: Administration of a priming dose of alfaxalone to healthy sedated cats reduced the total dose of alfaxalone needed to achieve orotracheal intubation, maintained mean arterial blood pressure, and did not adversely impact the measured respiratory variables.

Phytocystatins are plant cystatins that are related to several physiological processes regulating endogenous cysteine proteases involved in seed development and germination, programmed cell death and response to stress conditions. In addition, phytocystatins can act in plant defense against exogenous peptidases from herbivorous insects, pathogens and nematodes. Considering that Citrus fruits are important to human nutrition and represent a high value crop in worldwide agriculture, in the present work, we performed the identification of putative cystatins from Citrus sinensis and from Citrus clementine and submitted them to phylogenetic analysis. Six cystatins from each species were identified as orthologous and classified into three well supported phylogenetic groups. Five cystatins representative of the phylogenetic groups were recombinantly expressed and the in vitro studies revealed them to be potent inhibitors against the cysteine peptidases papain, legumain, human cathepsins (B, L, S, K) and a cathepsin B-like from Diaphorina citri (the Asian Citrus psyllid). Our findings provide the C. clementina and C. sinensis cystatins classification and an enzyme-inhibitor interactions profile, which may reflect an evolutionary process of Citrus cystatins related to gene functions as initial germination rates and seedlings development as well associated to plant defense against pathogens, as insects and nematodes., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest in this work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Serratia marcescens has emerged as an important opportunistic pathogen responsible for nosocomial and severe infections. Here, we determined phenotypic and molecular characteristics of 54 S. marcescens isolates obtained from patient samples from intensive-care-unit (ICU) and neonatal intensive-care-unit (NIUC) of a Brazilian tertiary hospital. All isolates were resistant to beta-lactam group antibiotics, and 92.6% (50/54) were not susceptible to tigecycline. Furthermore, 96.3% showed intrinsic resistance to polymyxin E (colistin), a last-resort antibiotic for the treatment of infections caused by MDR (multidrug-resistant) Gram-negative bacteria. In contrast, high susceptibility to other antibiotics such as fluoroquinolones (81.5%), and to aminoglycosides (as gentamicin 81.5%, and amikacin 85.2%) was found. Of all isolates, 24.1% were classified as MDR. The presence of resistance and virulence genes were examined by PCR and sequencing. All isolates carried KPC-carbapenemase ( bla KPC ) and extended spectrum beta-lactamase bla TEM genes, 14.8% carried bla OXA- 1 , and 16.7% carried bla CTX-M- 1 group genes, suggesting that bacterial resistance to β-lactam antibiotics found may be associated with these genes. The genes Sde B/ Has F and Sde Y/ Has F that are associated with efflux pump mediated drug extrusion to fluoroquinolones and tigecycline, respectively, were found in 88.9%. The aac(6')-Ib-cr variant gene that can simultaneously induce resistance to aminoglycoside and fluoroquinolone was present in 24.1% of the isolates. Notably, the virulence genes to (i) pore-forming toxin ( Shl A); (ii) phospholipase with hemolytic and cytolytic activities ( Phl A); (iii) flagellar transcriptional regulator ( Flh D); and (iv) positive regulator of prodigiosin and serratamolide production ( Pig P) were present in 98.2%. The genetic relationship among the isolates determined by ERIC-PCR demonstrated that the vast majority of isolates were grouped in a single cluster with 86.4% genetic similarity. In addition, many isolates showed 100% genetic similarity to each other, suggesting that the S. marcescens that circulate in this ICU are closely related. Our results suggest that the antimicrobial resistance to many drugs currently used to treat ICU and NIUC patients, associated with the high frequency of resistance and virulence genes is a worrisome phenomenon. Our findings emphasize the importance of active surveillance plans for infection control and to prevent dissemination of these strains., (Copyright © 2020 Ferreira, Rezende, Damas, Oliveira-Silva, Pitondo-Silva, Brito, Leonardecz, Góes, Campanini, Malavazi, da Cunha and Pranchevicius.)

Celiac disease (CD) is a chronic illness characterized by an inflammatory process triggered by gluten protein intake. Recent evidence has suggested that the lower relative abundance of bifidobacteria in the intestinal lumen may be associated with CD. Herein, we assessed the effect of the Bifidobacterium species Bifidobacterium bifidum , Bifidobacterium longum , Bembidion breve , Bifidobacterium animalis alone, and also a Bifidobacterium consortium on the digestion of intact gluten proteins (gliadins and glutenins) and the associated immunomodulatory responses elicited by the resulting peptides. The cytotoxicity and proinflammatory responses were evaluated through the activation of NF-kB p65 and the expression of cytokines TNF-α and IL-1β in Caco-2 cell cultures exposed to gluten-derived peptides. The peptides induced a clear reduction in cytotoxic responses and proinflammatory marker levels compared to the gluten fragments generated during noninoculated gastrointestinal digestion. These results highlight the possible use of probiotics based on bifidobacteria as a prospective treatment for CD.

Aspergillus fumigatus is a major cause of human disease. The survival of this fungus is dependent on the cell wall organization and function of its components. The cell wall integrity pathway (CWIP) is the primary signaling cascade that controls de novo synthesis of the cell wall in fungi. Abundant conidiation is a hallmark in A. fumigatus , and uptake of conidia by a susceptible host is usually the initial event in infection. The formation of conidia is mediated by the development of fungus-specific specialized structures, conidiophores, which are accompanied by cell wall remodeling. The molecular regulation of these changes in cell wall composition required for the rise of conidiophore from the solid surface and to disperse the conidia into the air is currently unknown. Here, we investigated the role of CWIP in conidiation. We show that CWIP pkcA G579R , Δ mpkA , and Δ rlmA mutants displayed reduced conidiation during synchronized asexual differentiation. The transcription factor RlmA directly regulated the expression of regulators of conidiation, including flbB , flbC , brlA , abaA , and rasB , as well as genes involved in cell wall synthesis and remodeling, and this affected the chitin content in aerial hyphae. Phosphorylation of RlmA and MpkA was increased during asexual differentiation. We also observed that MpkA physically associated with the proteins FlbB, FlbC, BrlA, and RasB during this process, suggesting another level of cross talk between the CWIP and asexual development pathways. In summary, our results support the conclusion that one function of the CWIP is the regulation of asexual development in filamentous fungi. IMPORTANCE A remarkable feature of the human pathogen Aspergillus fumigatus is its ability to produce impressive amounts of infectious propagules known as conidia. These particles reach immunocompromised patients and may initiate a life-threatening mycosis. The conidiation process in Aspergillus is governed by a sequence of proteins that coordinate the development of conidiophores. This process requires the remodeling of the cell wall so that the conidiophores can rise and withstand the chains of conidia. The events regulating cell wall remodeling during conidiation are currently unknown. Here, we show that the cell wall integrity pathway (CWIP) components RlmA and MpkA directly contribute to the activation of the conidiation cascade by enabling transcription or phosphorylation of critical proteins involved in asexual development. This study points to an essential role for the CWIP during conidiation and provides further insights into the complex regulation of asexual development in filamentous fungi., (Copyright © 2020 American Society for Microbiology.)

Objective: To compare results of a commercially available device for oscillometrically measured blood pressure (OBP) with invasively measured blood pressure (IBP) in awake and anesthetized dogs., Animals: 19 adult dogs (mean ± SD body weight, 17.8 ± 7.5 kg)., Procedures: Blood pressures were measured in dogs while they were awake and anesthetized with isoflurane. The OBP was recorded on a thoracic limb, and IBP was simultaneously recorded from the median caudal artery. Agreement between OBP and IBP was evaluated with the Bland-Altman method. Guidelines of the American College of Veterinary Internal Medicine (ACVIM) were used for validation of the oscillometric device., Results: In awake dogs, mean bias of the oscillometric device was -11.12 mm Hg (95% limits of agreement [LOA], -61.14 to 38.90 mm Hg) for systolic arterial blood pressure (SAP), 9.39 mm Hg (LOA, -28.26 to 47.04 mm Hg) for diastolic arterial blood pressure (DAP), and -0.85 mm Hg (LOA, -40.54 to 38.84 mm Hg) for mean arterial blood pressure (MAP). In anesthetized dogs, mean bias was -12.27 mm Hg (LOA, -47.36 to 22.82 mm Hg) for SAP, -3.92 mm Hg (LOA, -25.28 to 17.44 mm Hg) for DAP, and -7.89 mm Hg (LOA, -32.31 to 16.53 mm Hg) for MAP. The oscillometric device did not fulfill ACVIM guidelines for the validation of such devices., Conclusions and Clinical Relevance: Agreement between OBP and IBP results for awake and anesthetized dogs was poor. The oscillometric blood pressure device did not fulfill ACVIM guidelines for validation. Therefore, clinical use of this device cannot be recommended.

Beta-thalassaemia (BT) is classified according to blood transfusion requirement as minor (BTMi), intermedia (BTI) and major (BTM). BTM is the most severe form, requiring regular transfusions while transfusion need is only occasional in BTI. Differential gene expression between patients has not been assessed so far. Here, we evaluated the global gene expression profiles during differentiation of human erythroid cells of two patients carrying the same mutation [CD39, (C → T)], though displaying different phenotypes (BTI and BTM). Considering the role of reactive oxygen species (ROS) in the pathophysiology of thalassaemia, we focused on differentially expressed genes involved in metabolic pathways triggered by ROS, such as inflammation and apoptosis, and, from these, we selected the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and High Mobility Group Box1 (HMGB1) genes, whose role in BT is not well established. An in-depth expression analysis of transcriptional and protein levels in patients carrying a range of mutations associated with BT phenotypes indicated that APEX1 was increased in both BTI and BTM. Furthermore, higher amounts of HMGB1 was found in the plasma of BTI patients. Our findings suggest that these proteins have important roles in BT and could represent new targets for further studies aiming to improve the management of the disease., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

Klebsiella pneumoniae is an important opportunistic pathogen that commonly causes nosocomial infections and contributes to substantial morbidity and mortality. We sought to investigate the antibiotic resistance profile, pathogenic potential and the clonal relationships between K. pneumoniae ( n = 25) isolated from patients and sources at a tertiary care hospital's intensive care units (ICUs) in the northern region of Brazil. Most of K. pneumoniae isolates ( n = 21, 84%) were classified as multidrug resistant (MDR) with high-level resistance to β-lactams, aminoglycosides, quinolones, tigecycline, and colistin. All the 25 isolates presented extended-spectrum beta-lactamase-producing (ESBL), including carbapenemase producers, and carried the bla KPC (100%), bla TEM (100%), bla SHV variants ( n = 24, 96%), bla OXA-1 group ( n = 21, 84%) and bla CTX-M-1 group ( n = 18, 72%) genes. The K2 serotype was found in 4% ( n = 1) of the isolates, and the K1 was not detected. The virulence-associated genes found among the 25 isolates were mrk D ( n = 24, 96%), fim H-1 ( n = 22, 88%), ent B (100%), iut A ( n = 10, 40%), ybt S ( n = 15, 60%). The genes related with efflux pumps and outer membrane porins found were AcrAB (100%), tol C ( n = 24, 96%), mdt K ( n = 22, 88%), Omp K35 ( n = 15, 60%), and Omp K36 ( n = 7, 28%). ERIC-PCR was employed to determine the clonal relationship between the different isolated strains. The obtained ERIC-PCR patterns revealed that the similarity between isolates was above 70%. To determine the sequence types (STs) a multilocus sequence typing (MLST) assay was used. The results indicated the presence of high-risk international clones among the isolates. In our study, the wide variety of MDR K. pneumoniae harboring β-lactams and virulence genes strongly suggest a necessity for the implementation of effective strategies to prevent and control the spread of antibiotic resistant infections.

Sphingolipids (SL) are complex lipids and components of the plasma membrane which are involved in numerous cellular processes, as well as important for virulence of different fungal pathogens. In yeast, SL biosynthesis is regulated by the "AGC kinases" Ypk1 and Ypk2, which also seem to connect the SL biosynthesis with the cell wall integrity (CWI) and the High Osmolarity Glycerol (HOG) pathways. Here, we investigate the role of ypkA Y PK1 in SL biosynthesis and its relationship with the CWI and the HOG pathways in the opportunistic human pathogen Aspergillus fumigatus . We found that ypkA is important for fungal viability, since the Δ ypkA strain presented a drastically sick phenotype and complete absence of conidiation. We observed that under repressive condition, the conditional mutant niiA::ypkA exhibited vegetative growth defects, impaired germination and thermosensitivity. In addition, the ypkA loss of function caused a decrease in glycosphingolipid (GSL) levels, especially the metabolic intermediates belonging to the neutral GSL branch including dihydroceramide (DHC), ceramide (Cer), and glucosylceramide (GlcCer), but interestingly a small increase in ergosterol content. Genetic analyzes showed that ypkA genetically interacts with the MAP kinases of CWI and HOG pathways, mpkA and sakA , respectively, while only SakA physically interacts with YpkA. Our results suggest that YpkA is important for fungal survival through the regulation of GSL biosynthesis and cross talks with A. fumigatus MAP kinase pathways.

Standing among the front defense strategies against pathogens, host phagocytic cells release various oxidants. Therefore, pathogens have to cope with stressful conditions at the site of infection. Peroxiredoxins (Prx) are highly reactive and abundant peroxidases that can support virulence and persistence of pathogens in distinct hosts. Here, we revealed that the opportunistic human pathogen A. fumigatus presents three 1-Cys Prx (Prx6 subfamily), which is unprecedented. We showed that PrxB and PrxC were in mitochondria, while Prx1 was in cytosol. As observed for other Prxs, recombinant Prx1 and PrxC decomposed H 2 O 2 at elevated velocities (rate constants in the 10 7  M -1 s -1 range). Deletion mutants for each Prx displayed higher sensitivity to oxidative challenge in comparison with the wild-type strain. Additionally, cytosolic Prx1 was important for A. fumigatus survival upon electron transport dysfunction. Expression of Prxs was dependent on the SakA HOG1 MAP kinase and the Yap1 YAP1 transcription factor, a global regulator of the oxidative stress response in fungi. Finally, cytosolic Prx1 played a major role in pathogenicity, since it is required for full virulence, using a neutropenic mouse infection model. Our data indicate that the three 1-Cys Prxs act together to maintain the redox balance of A. fumigatus.

Salmonellosis is a poultry industry and public health concern worldwide. Recently, Salmonella enterica serovar Heidelberg (SH) has been reported in broilers in Brazil. The effect of feeding a blend of three strains of Bacillus subtilis (PRO) was studied in broilers orally challenged (10 7 CFU/chick) or not with a SH isolated in south of Brazil (UFPR1 strain). Twelve male Cobb 500 broilers per pen were randomly assigned to six treatments in a 3 × 2 factorial experiment where PRO was added at 0, 250, or 500 g/ton of broiler feed and fed to either SH-challenged (SH Control, SH + PRO 250, and SH + PRO 500) or non-challenged birds (Control, PRO 250, and PRO 500). Broiler performance, histologic alterations in intestinal morphology, Salmonella quantification and immune cells counts in liver (macrophages, T CD4+ and T CD8+) were analyzed. Changes in the intestinal microbiota of broilers were also studied by metagenomics for Control, SH Control, SH + PRO 250, and SH + PRO 500 only. Feeding PRO at 250 or 500 g/ton reduced SH counts and incidence in liver and cecum at 21 days of age. It was observed that PRO groups increased the macrophage mobilization to the liver in SH-challenged birds ( P  < 0.05) but reduced these cells in the liver of non-challenged birds, showing an interesting immune cell dynamics effect. PRO at 250 g/ton did not affect gut histology, but improved animal performance ( P  < 0.05) while PRO at 500/ton did not affect animal performance but increased histologic alteration related to activation of the defense response in the ileum in SH challenged birds compared to control birds ( P  < 0.05). SH + PRO 500 group presented a more diverse cecal microbiota (Shannon-Wiener index; P  < 0.05) compared to Control and SH Control groups; while SH + PRO 250 had greater ileal richness (JackkNife index) compared to Control ( P  < 0.05). PRO was effective in reducing Salmonella colonization in liver and cecum when fed at 250 or 500 g/ton to broilers inoculated with SH strain UFPR1. PRO promotes positive alterations in performance (at 250 g/ton), immune modulatory effect in the gastrointestinal tract, SH reduction, and intestinal microbiota modulation.

Vaccinia virus (VACV) is the agent of bovine vaccinia (BV), an emerging zoonosis that causes exanthematic lesions on the teats of dairy cows and on the hands of milkers. The virus has been detected in the milk of naturally infected cows. The objective of this study was to investigate and quantify VACV DNA as well as the presence of infectious virus particles in samples of cheese curd, cheese whey and pasteurized milk produced using milk from cows experimentally inoculated with VACV-GP2, a Brazilian isolate of VACV (VACV-BR). VACV DNA was detected in samples of cheese and pasteurized milk at different time points, even after the resolution of the typical lesions caused by VACV, which occurred after 22 days post-infection (dpi), on average. Moreover, it was possible to detect infectious viral particles in cheese samples on alternate days until 27 dpi. The presence of both VACV DNA and infectious viral particles in cheese samples throughout the clinical course of BV and even after the disappearance of the typical clinical signs of disease draws attention to the risk associated with consumption of the cheese. Furthermore, VACV-contaminated milk and cheese may represent an occupational risk to cheesemakers who often manipulate milk and cheese curd without wearing gloves., (© 2017 Blackwell Verlag GmbH.)

Salmonella enterica serovar Heidelberg is a human pathogen also found in broilers. A strain (UFPR1) has been associated with field reports of resistance to short-chain organic acids (SCOA) in broilers in the South of Brazil, but was susceptible to a Bacillus subtilis -based probiotic added in feed in a related study. This work aimed to (i) report clinical symptoms caused by SH UFPR1 in broilers, (ii) study its susceptibility to some antibiotics in vitro , and (iii) SCOA in vivo ; and (iv) relate these phenotypic observations with its genome characteristics. Two in vivo trials used 1-day-old chicks housed for 21 days in 8 sterilized isolated negative pressure rooms with 4 battery cages of 12 birds each. Birds were challenged or not with 10 7  CFU/bird of SH UFPR1 orally and exposed or not to SCOA in a 2 × 2 factorial design. Zootechnical parameters were unaffected ( P  > 0.05), no clinical signs were observed, and few cecal and hepatic histologic and immune-related alterations were seen, in birds challenged with SH. Formic and propionic acids added together in drinking water, fumaric and benzoic acid in feed (Trial 1), and coated calcium butyrate in feed (Trial 2) did not reduce the SH isolation frequencies seen in cecum and liver in broilers after SH challenge ( P  > 0.05). SH UFPR1 was susceptible to amikacin, amoxicillin + clavulanate, ceftiofur, cephalexin, doxycycline and oxytetracycline; and mildly susceptible to ampicillin + sulbactam, cephalothin, ciprofloxacin, enrofloxacin, and gentamycin in an in vitro minimum inhibitory concentration model using Mueller-Hinton agar. The whole genome of SH UFPR1 was sequenced and consisted of a circular chromosome, spanning 4,760,321 bp with 52.18% of GC-content encoding 84 tRNA, 22 rRNA, and 4,427 protein-coding genes. The comparison between SH UFPR1 genome and a multidrug-resistant SL476 strain revealed 11 missing genomic fragments and 5 insertions related to bgt, bgr , and rpoS genes. The deleted genes codify proteins associated with cell cycle regulation, virulence, drug resistance, cellular adhesion, and salt efflux which collectively reveal key aspects of the evolution and adaptation of SH strains such as organic acids resistance and antibiotic sensitivity and provide information relevant to the control of SH in poultry.

Objectives: 1) To determine which peripheral artery commonly used for invasive arterial blood pressure (IBP) monitoring yields the least bias when compared with noninvasive blood pressure (NIBP) values obtained at the antebrachium of the dog, and 2) to identify and describe differences in systolic (SAP), mean (MAP) and diastolic arterial pressures (DAP) among different anatomical locations., Study Design: Prospective experimental study., Animals: Twenty adult hound dogs weighing 24.5 ± 1.1 kg (mean ± standard deviation)., Methods: Four peripheral arteries-dorsal pedal, median caudal, intermediate auricular and superficial palmar arteries-were catheterized with 20 gauge, 3.8 cm catheters. One NIBP cuff was placed in the middle third of the antebrachium. Four sets of IBP and NIBP measurements were simultaneously collected every 2 minutes. A linear mixed model was performed to analyze the collected data., Results: IBP values varied depending on the arterial catheterization site. The difference was greater for SAP. NIBP measured at the antebrachium had the best agreement with IBP measured at the median caudal artery., Conclusion and Clinical Relevance: IBP varies among anatomical locations. The smallest bias and narrowest limits of agreement were obtained at the median caudal artery, providing the best overall agreement with the equipment studied. The median caudal artery may be the preferable anatomical location for clinical comparison studies between IBP and NIBP in dogs when the cuff is on the antebrachium., (Published by Elsevier Ltd.)

A 3-month-old male umbrella cockatoo (Cacatua alba) was presented because of acute non-weight-bearing lameness of the right leg. Marked soft tissue swelling was present around the femorotibiotarsal (stifle) joint, and the radiographic diagnosis was right medial femorotibiotarsal subluxation. Surgical management was elected, and the stifle joint was approached via a lateral parapatellar incision. Joint exploration revealed damage to the lateral meniscus, tendon of origin of the cranial tibial muscle, and cranial cruciate ligament. After debriding the disrupted meniscus, the stifle joint was anatomically reduced. The femorotibiotarsal joint was stabilized by using a lateral extracapsular suture in a modified technique using a self-tapping cortical screw in the lateral femoral condyle and a hole through the proximal tibiotarsus. The bird regained function of the femorotibiotarsal joint for 3 months after surgery, allowing sufficient time for the bird to establish a physiologic perching angle so that ankylosis occurred to maintain functionality of the leg as a unit. This combination of orthopedic techniques adapted from techniques commonly used in small companion-animal species may be considered to provide young birds with femorotibiotarsal luxations and subluxation a good quality of life despite ankylosis of the joint.

OBJECTIVE To evaluate agreement among diplomates of the American College of Veterinary Anesthesia and Analgesia for scores determined by use of a simple descriptive scale (SDS) or a composite grading scale (CGS) for quality of recovery of horses from anesthesia and to investigate use of 3-axis accelerometry (3AA) for objective evaluation of recovery. ANIMALS 12 healthy adult horses. PROCEDURES Horses were fitted with a 3AA device and then were anesthetized. Eight diplomates evaluated recovery by use of an SDS, and 7 other diplomates evaluated recovery by use of a CGS. Agreement was tested with κ and AC1 statistics for the SDS and an ANOVA for the CGS. A library of mathematical models was used to map 3AA data against CGS scores. RESULTS Agreement among diplomates using the SDS was slight (κ = 0.19; AC1 = 0.22). The CGS scores differed significantly among diplomates. Best fit of 3AA data against CGS scores yielded the following equation: RS = 9.998 × SG 0.633 × ∑UG 0.174 , where RS is a horse's recovery score determined with 3AA, SG is acceleration of the successful attempt to stand, and ∑UG is the sum of accelerations of unsuccessful attempts to stand. CONCLUSIONS AND CLINICAL RELEVANCE Subjective scoring of recovery of horses from anesthesia resulted in poor agreement among diplomates. Subjective scoring may lead to differences in conclusions about recovery quality; thus, there is a need for an objective scoring method. The 3AA system removed subjective bias in evaluations of recovery of horses and warrants further study.

Objective: To determine the median effective dose (ED 50 ) of intravenous (IV) bupivacaine associated with a 50% probability of causing clinically relevant cardiovascular effects [defined as 30% change in heart rate (HR) or mean arterial pressure (MAP)] in chickens anesthetized with isoflurane., Study Design: Randomized up-and-down study., Animals: A total of 14 Ross-708 broiler chickens (Gallus gallus domesticus) weighing 1.70-2.75 kg., Methods: Anesthesia was induced and maintained with isoflurane. Monitoring included the electrocardiogram and invasive arterial pressures. Chickens were administered bupivacaine IV over 2 minutes using a dose based on the response of the previous animal. Dose was decreased when HR and/or MAP in the previous animal increased or decreased ≥30% after bupivacaine administration, or increased when HR or MAP changed <30%. The ED 50 was defined as the dose resulting in ≥30% variation in HR or MAP in 50% of the population studied., Results: The IV ED 50 of bupivacaine was 1.94 mg kg -1 using Dixon's up-and-down method and 1.96 mg kg -1 by logistic regression., Conclusions and Clinical Relevance: These results suggest that 1.33 and 1.96 mg kg -1 of IV bupivacaine are associated with a respective 1 or 50% probability of a clinically significant change in MAP in isoflurane-anesthetized chickens. Identification of the cardiovascular changes associated with different doses of bupivacaine can be used as the basis for studies of therapeutic applications in the domestic chicken. Further studies are required to determine interspecies variation., (Published by Elsevier Ltd.)

This study evaluated the effects of the Biosilicate ® and poly (D,L-lactic-co-glycolic) acid composites on bone repair in a tibial bone defect model in rats by means of using histological evaluation (histopathological and morphometric analysis) and gene expression analysis. Eighty male Wistar rats (12 weeks old, weighing ±300 g) were randomly divided into two groups: Biosilicate ® group (BG) and Biosilicate ® /PLGA group (BG/PLGA). Each group was euthanized at 3, 7, 14, and 21 days after surgery (n = 10 animals per time point). The main findings showed that the incorporation of PLGA into BG had a significant effect on the morphological structure of the material, accelerating mass loss, decreasing the pH and increasing the calcium release. Furthermore, histologic analysis revealed that the BG/PLGA showed increased material degradation, accompanied by higher bone formation compared to BG, after 21 days of implantation. In addition, qRT-PCR analysis showed that BG/PLGA induced an upregulation of the osteogenic genes related to BMP4, Runx2, ALP, and OC. These results show that the present BG/PLGA composite may be used as a bone graft for inducing bone repair. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 63-71, 2017., (© 2015 Wiley Periodicals, Inc.)

This study investigated the effects of ketamine and lidocaine in combination on the minimum alveolar concentration of sevoflurane (MACSEVO) in alpacas. Eight healthy, intact male, adult alpacas were studied on 2 separate occasions. Anesthesia was induced with SEVO, and baseline MAC (MACB) determination began 45 min after induction. After MACB determination, alpacas were randomly given either an intravenous (IV) loading dose (LD) and infusion of saline or a loading dose [ketamine = 0.5 mg/kg body weight (BW); lidocaine = 2 mg/kg BW] and an infusion of ketamine (25 μg/kg BW per minute) in combination with lidocaine (50 μg/kg BW per minute), and MACSEVO was re-determined (MACT). Quality of recovery, time-to-extubation, and time-to-standing, were also evaluated. Mean MACB was 1.88% ± 0.13% and 1.89% ± 0.14% for the saline and ketamine + lidocaine groups, respectively. Ketamine and lidocaine administration decreased (P < 0.05) MACB by 57% and mean MACT was 0.83% ± 0.10%. Saline administration did not change MACB. Time to determine MACB and MACT was not significantly different between the treatments. The quality of recovery, time-to-extubation, and time-to-standing, were not different between groups. The infusion of ketamine combined with lidocaine significantly decreased MACSEVO by 57% and did not adversely affect time-to-standing or quality of recovery.

Objectives: To compare two commonly used oscillometric technologies for obtaining noninvasive blood pressure (NIBP) measurements and to determine if there is a difference in agreement between these systems and invasive blood pressure (IBP) measurements., Study Design: Prospective, experimental study., Animals: Twenty adult laboratory dogs., Methods: Each dog was anesthetized and its median caudal artery catheterized for IBP monitoring. An NIBP cuff was placed in the middle third of the antebrachium and attached to either monitor-1 or monitor-2. Four pairs of concurrent NIBP and IBP measurements were recorded with each monitor. Agreement between IBP and NIBP measurements was explored using Bland-Altman analysis, as well as the American College of Veterinary Internal Medicine (ACVIM) and Association for the Advancement of Medical Instrumentation (AAMI) guidelines for the validation of NIBP devices., Results: Both NIBP technologies produced results that met the ACVIM and AAMI guidelines for the validation of NIBP devices. For monitor-1, analyses of agreement showed biases of 0.2 mmHg [95% limits of agreement (LoA) -11.8 to 12.3 mmHg] in systolic arterial pressure (SAP) values, -2.6 mmHg (95% LoA -14.4 to 9.1 mmHg) in diastolic arterial pressure (DAP) values, and -2.5 mmHg (95% LoA -12.7 to 7.3 mmHg) in mean arterial pressure (MAP) values. For monitor-2, analyses of agreement showed biases of 3.4 mmHg (95% LoA -8.7 to 15.5 mmHg) in SAP values, 2.2 mmHg (95% LoA -6.6 to 10.9 mmHg) in DAP values, and 1.6 mmHg (95% LoA -5.9 to 8.9 mmHg) in MAP values., Conclusions and Clinical Relevance: Multi-function monitors can contain components from various manufacturers. Clinicians should consider whether these have been validated in the species to be monitored. Both of the technologies studied here seem appropriate for use in dogs., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Objective: To compare blood pressure measured noninvasively with an oscillometric device that involved use of a novel conical cuff and a traditional cylindrical blood pressure cuff., Animals: 17 adult hound-type dogs., Procedures: Dogs were anesthetized, and a 20-gauge, 1.5-inch catheter was inserted in the median sacral artery. The catheter was attached to a pressure transducer via fluid-filled noncompliant tubing, and direct blood pressure was recorded with a multifunction monitor. A specially fabricated conical cuff was placed on the antebrachium. Four sets of direct and indirect blood pressure measurements were simultaneously collected every 2 minutes. Four sets of measurements were then obtained by use of a cylindrical cuff., Results: The cylindrical cuff met American College of Veterinary Internal Medicine consensus guidelines for validation of indirect blood pressure measurements for mean arterial blood pressure (MAP), systolic arterial blood pressure (SAP), and diastolic arterial blood pressure (DAP). The conical cuff met the consensus guidelines for difference of paired measurements, SD, and percentages of measurements within 10 and 20 mm Hg of the value for the reference method, but it failed a correlation analysis. In addition, although bias for the conical cuff was less than that for the cylindrical cuff for SAP, MAP, and DAP measurements, the limits of agreement for the conical cuff were wider than those for the cylindrical cuff for SAP and MAP measurements., Conclusions and Clinical Relevance: On the basis of results of this study, use of a conical cuff for oscillometric blood pressure measurement cannot be recommended.

The objective was to compare plasma lidocaine concentrations when a commercially available 5% lidocaine patch was placed on intact skin vs. an incision. Our hypothesis was that greater absorption of lidocaine would occur from the incision site compared to intact skin. Ten dogs were used in a crossover design. A patch was placed over an incision, and then after a washout period, a patch was placed over intact skin. Plasma lidocaine concentrations were measured at patch placement; 20, 40 and 60 min; and 2, 4, 6, 12, 24, 36, 48, 72 and 96 h after patch placement. After patch removal, the skin was graded using a subjective skin reaction system. No dogs required rescue analgesia, and no toxicity or skin reaction was noted. Mean ± SD AUC and CMAX were 3054.29 ± 1095.93 ng·h/mL and 54.1 ± 15.84 ng/mL in the Incision Group, and 2269.9 ± 1037.08 ng·h/mL and 44.5 ± 16.34 ng/mL in the No-Incision Group, respectively. The AUC was significantly higher in the Incision Group. The results of the study demonstrate that the actual body exposure to lidocaine was significantly higher when an incision was present compared to intact skin. No adverse effects were observed from either treatment. Efficacy was not evaluated., (© 2015 John Wiley & Sons Ltd.)

Objective: To determine the cardiovascular effects of lidocaine infused intravenously (IV) in broiler chickens., Study Design: Two phase study: Phase 1, randomized up-and-down study to determine effective dose 50 (ED50) for lidocaine; Phase 2, prospective randomized study to determine the cardiovascular effects of lidocaine., Animals: Seventeen Ross-708 broiler chickens (Gallus gallus domesticus) [11 chickens (Phase 1) and 6 chickens (Phase 2)], weighing 2.6-4.3 kg., Methods: After induction of anesthesia with isoflurane and placement of monitoring equipment including invasive blood pressure, chickens were administered lidocaine IV. During Phase 1, using an up-and-down design, each animal received a variable dose selected based on the response of the previous animal. During Phase 2, each animal was administered 6 mg kg(-1) of lidocaine IV over 2 minutes. Clinically irrelevant cardiovascular effects were defined as a relative decrease of heart rate (HR) and mean blood pressure (MAP) <30% subsequent to IV lidocaine administration. The ED50 was defined as the dose rate that would cause clinically irrelevant cardiovascular depression in 50% of the population., Results: During Phase 1, using an up-and-down study design (n = 11), the ED50 of lidocaine was determined to be 6.30 mg kg(-1) and 6.22 mg kg(-1) (95% confidence interval, 5.30-7.13 mg kg(-1)), when calculated by Dixon's up-and-down method, and logistic regression, respectively. During Phase 2, following infusion of lidocaine (6 mg kg(-1)), no clinically relevant effects on HR or MAP were detected in any animal., Conclusions and Clinical Relevance: Previous reports state that the dose of lidocaine used in birds should be ≤4 mg kg(-1). In this study, 6 mg kg(-1) of lidocaine injected IV was not associated with adverse cardiovascular effects. These results suggest that the dose of 4 mg kg(-1) can be exceeded, at least in chickens, and opens the possibility of other therapeutic uses for lidocaine in birds., (© 2014 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia.)

Objective: To determine whether directly measured arterial blood pressure differs among anatomic locations and whether arterial blood pressure is influenced by body position., Animals: 33 client-owned dogs undergoing anesthesia., Procedures: Dogs undergoing anesthetic procedures had 20-gauge catheters placed in both the superficial palmar arch and the contralateral dorsal pedal artery (group 1 [n = 20]) or the superficial palmar arch and median sacral artery (group 2 [13]). Dogs were positioned in dorsal recumbency, and mean arterial blood pressure (MAP), systolic arterial blood pressure (SAP), and diastolic arterial blood pressure (DAP) were recorded for both arteries 4 times (2-minute interval between successive measurements). Dogs were positioned in right lateral recumbency, and blood pressure measurements were repeated., Results: Differences were detected between pressures measured at the 2 arterial sites in both groups. This was especially true for SAP measurements in group 1, in which hind limb measurements were a mean of 16.12 mm Hg higher than carpus measurements when dogs were in dorsal recumbency and 14.70 mm Hg higher than carpus measurements when dogs were in lateral recumbency. Also, there was significant dispersion about the mean for all SAP, DAP, and MAP measurements., Conclusions and Clinical Relevance: Results suggested that arterial blood pressures may be dependent on anatomic location and body position. Because this may affect outcomes of studies conducted to validate indirect blood pressure measurement systems, care must be used when developing future studies or interpreting previous results.