The Resistance of Bacillus Spores: Implications for the Strain-Specific Response to High-Performance Disinfectants.

Bacterial spores in materials and equipment pose significant biosecurity risks, making effective disinfection crucial. This study evaluated Ortho-phthalaldehyde (OPA) and a quaternary ammonia-glutaraldehyde solution (AG) for inactivating spores of Bacillus thuringiensis (BT), B. cereus (BC), and two strains of B. velezensis (BV1 and BV2). Spores of BV1 and BT were treated with 22.5 mg/m3 OPA by dry fumigation or 1 mg/mL AG by spray for 20 min, according to the manufacturer's recommendation. As no sporicidal effect was observed, OPA was tested at 112.5 mg/m3 for 40 min, showing effectiveness for BT but not for BV1. Minimum bactericidal concentration (MBC) tests revealed higher MBC values for glutaraldehyde, prompting an overnight test with 112.5 mg/m3 OPA by dry fumigation and 50 mg/mL AG by spray, using formaldehyde as a control. AG reduced all Bacillus strains, but with limited sporicidal effect. OPA was sporicidal for BT and BV1 but not for BC and BV2, indicating a strain-dependent effect. Formaldehyde performed better overall but did not completely inactivate BV2 spores. Our findings suggest that OPA and AG have potential as formaldehyde replacements in wet disinfection procedures.

A new standardization for the use of chicken embryo: selection of target from the phage display library and infection.

The filamentous bacteriophage M13KO7 (M13) is the most used in phage display (PD) technology and, like other phages, has been applied in several areas of medicine, agriculture, and in the food industry. One of the advantages is that they can modulate the immune response in the presence of pathogenic microorganisms, such as bacteria and viruses. This study evaluated the use of phage M13 in the chicken embryos model. We inoculated 13-day-old chicken embryos with Salmonella Pullorum (SP) and then evaluated survival for the presence of phage M13 or E. coli ER2738 (ECR) infected with M13. We found that the ECR bacterium inhibits SP multiplication in 0.32 (M13-infected ECR) or 0.44 log UFC/mL (M13-uninfected ECR) and that the ECR-free phage M13 from the PD library can be used in chicken embryo models. This work provides the use of the chicken embryo as a model to study systemic infection and can be employed as an analysis tool for various peptides that M13 can express from PD selection. KEY POINTS: • SP-infected chicken embryo can be a helpful model of systemic infection for different tests. • Phage M13 does not lead to embryonic mortality or cause serious injury to embryos. • Phage M13 from the PD library can be used in chicken embryo model tests.

Antimicrobial Activity of Essential-Oil-Based Nanostructured Lipid Carriers against Campylobacter Spp. Isolated from Chicken Carcasses.

Campylobacter is a virulent Gram-negative bacterial genus mainly found in the intestines of poultry. The indiscriminate use of traditional antibiotics has led to drug resistance in these pathogens, necessitating the development of more efficient and less toxic therapies. Despite their complex biologically active structures, the clinical applications of essential oils (EOs) remain limited. Therefore, this study aimed to increase the bioavailability, stability, and biocompatibility and decrease the photodegradation and toxicity of EO using nanotechnology. The diffusion disk test revealed the potent anti-Campylobacter activity of cinnamon, lemongrass, clove, geranium, and oregano EOs (>50 mm). These were subsequently used to prepare nanostructured lipid carriers (NLCs). Formulations containing these EOs inhibited Campylobacter spp. growth at low concentrations (0.2 mg/mL). The particle size, polydispersity index, and zeta potential of these systems were monitored, confirming its physicochemical stability for 210 days at 25 °C. FTIR-ATR and DSC analyses confirmed excellent miscibility among the excipients, and FE-SEM elucidated a spherical shape with well-delimited contours of nanoparticles. The best NLCs were tested regarding nanotoxicity in a chicken embryo model. These results indicate that the NLC-based geranium EO is the most promising and safe system for the control and treatment of multidrug-resistant strains of Campylobacter spp.

Five centuries of genome evolution and multi-host adaptation of Campylobacter jejuni in Brazil.

Consumption of raw, undercooked or contaminated animal food products is a frequent cause of Campylobacter jejuni infection. Brazil is the world's third largest producer and a major exporter of chicken meat, yet population-level genomic investigations of C. jejuni in the country remain scarce. Analysis of 221 C. jejuni genomes from Brazil shows that the overall core and accessory genomic features of C. jejuni are influenced by the identity of the human or animal source. Of the 60 sequence types detected, ST353 is the most prevalent and consists of samples from chicken and human sources. Notably, we identified the presence of diverse bla genes from the OXA-61 and OXA-184 families that confer beta-lactam resistance as well as the operon cmeABCR related to multidrug efflux pump, which contributes to resistance against tetracyclines, macrolides and quinolones. Based on limited data, we estimated the most recent common ancestor of ST353 to the late 1500s, coinciding with the time the Portuguese first arrived in Brazil and introduced domesticated chickens into the country. We identified at least two instances of ancestral chicken-to-human infections in ST353. The evolution of C. jejuni in Brazil was driven by the confluence of clinically relevant genetic elements, multi-host adaptation and clonal population growth that coincided with major socio-economic changes in poultry farming.

Harnessing probiotics capability to combat Salmonella Heidelberg and improve intestinal health in broilers.

The poultry industry faces significant challenges in controlling Salmonella contamination while reducing antibiotic use, particularly with the emergence of Salmonella Heidelberg (SH) strains posing risks to food safety and public health. Probiotics, notably lactic acid bacteria (LAB) and Saccharomyces boulardii (SB) offer promising alternatives for mitigating Salmonella colonization in broilers. Understanding the efficacy of probiotics in combating SH and their impact on gut health and metabolism is crucial for improving poultry production practices and ensuring food safety standards. This study aimed to assess the inhibitory effects of LAB and SB against SH both in vitro and in vivo broilers, while also investigating their impact on fecal metabolites and caecal microbiome composition. In vitro analysis demonstrated strong inhibition of SH by certain probiotic strains, such as Lactiplantibacillus plantarum (LP) and Lacticaseibacillus acidophilus (LA), while others like SB and Lactobacillus delbrueckii (LD) did not exhibit significant inhibition. In vivo testing revealed that broilers receiving probiotics had significantly lower SH concentrations in cecal content compared to the positive control (PC) at all ages, indicating a protective effect of probiotics against SH colonization. Metagenomic analysis of cecal-content microbiota identified predominant bacterial families and genera, highlighting changes in microbiota composition with age and probiotic supplementation. Additionally, fecal metabolomics profiling showed alterations in metabolite concentrations, suggesting reduced oxidative stress, intestinal inflammation, and improved gut health in probiotic-supplemented birds. These findings underscore the potential of probiotics to mitigate SH colonization and improve broiler health while reducing reliance on antibiotics.

Antitumor activity of essential oils-based nanostructured lipid carriers on prostate cancer cells.

Prostate cancer (PCa) is the second most frequent malignancy in men worldwide. Essential oils (EOs) are natural products which can act in cancer suppression by several mechanisms. In this work, a nanotechnological approach was used to develop and evaluate the antineoplastic effects of EOs loaded by nanostructured lipid carriers (NLCs). Three different NLC systems composed of cinnamon, sage or thyme EOs were optimized using factorial design (23). The optimal formulations were characterized in terms of biophysical parameters, structure, stability, in vivo safety and efficacy. All optimized NLC formulations exhibited excellent structural properties and stability over a year (25 °C). They proved to be in vitro and in vivo biocompatible on PNT2 normal prostate cells and on chicken embryos (CE), respectively. In PC3 PCa cells, optimized NLCs inhibited cell proliferation and migration and changed its morphology. In CE xenograft tumor, NLCs have inhibited tumor growth and angiogenesis. The results from this work suggested that all developed EO-based NLC formulations had their stability improved while the biological activity remains unchanged.

Unlocking the power of Libidibia ferrea extracts: antimicrobial, antioxidant, and protective properties for potential use in poultry production.

Plant extracts are increasingly recognized as potential prophylactic agents in poultry production due to their diverse bioactive properties. This study investigated the phytochemical and biological properties of Libidibia ferrea (L. ferrea), a plant species native to the Caatinga region of northeastern Brazil. The aim of this study was to identify secondary metabolites and to demonstrate the antimicrobial, antioxidant and protective effects of the plant extract. Three extracts were produced: EHMV, a hydroalcoholic extract from the maceration of pods, and EEMC and EEMV ethanolic extracts from the maceration of peels and pods, respectively, from L. ferrea. High-performance liquid chromatography (HPLC-MS/MS) and atomic absorption spectroscopy (AAS) were used to characterize the metabolites and metals. The antimicrobial activity against Salmonella Galinarum (SG), Salmonella pullorum (SP), Salmonella Heidelberg (SH) and Avian pathogenic Escherichia coli (APEC) was evaluated alone and in combination with probiotic bacteria (Bacillus velenzensis) using agar diffusion and the bactericidal minimum concentration (CBM). The antioxidant potential of the extracts was evaluated in 5 in vitro assays and 6 assays in 3t3 cells. The toxicity of EHMV was tested, and its ability to combat SP infection was demonstrated using a chicken embryo model. The results showed that EHMV exhibited significant antimicrobial activity. The combination of EHMV with BV had synergistic effects, increased antimicrobial activity and induced bacterial sporulation. Composition analysis revealed the presence of 8 compounds, including tannins and phenolic compounds. In vitro antioxidant tests demonstrated that total antioxidant capacity(TAC) activity was increased, and the extract had strong reducing power and notable metal chelating effects. Analysis of 3T3 cells confirmed the protective effect of EHMV against oxidative stress. Toxicity assessments in chicken embryos confirmed the safety of EHMV and its protective effect against SP-induced mortality. EHMV from L. ferrea is rich in proteins and contains essential metabolites that contribute to its antimicrobial and antioxidant properties. When associated with probiotic bacteria such as B. velezensis, this extract increases the inhibition of SH, SG, SP, and APE. The nontoxic nature of EHMV and its protective effects on chicken embryos make it a potential supplement for poultry.

Curcumin-functionalized gold nanoparticles attenuate AAPH-induced acute cardiotoxicity via reduction of lipid peroxidation and modulation of antioxidant parameters in a chicken embryo model.

Gold nanoparticles (AuNPs) have gained considerable attention due to their biocompatibility, customizable optical properties and ease of synthesis. In this study, an environmentally friendly method was used for synthesize curcumin-functionalized AuNPs (AuNP-C). AuNP-C exhibited a spherical shape, uniformity, and an average diameter of 6 nm. The in vitro antioxidant activity was analyzed, and cytotoxicity properties of AuNP-C were assessed in fibroblast and macrophage cells. Additionally, the effects of AuNP-C on oxidative stress in chicken embryo liver and hearts were investigated. AuNP-C demonstrated potent free radical scavenging properties without exhibiting cytotoxicity and hepatotoxicity effects. Administration of 300 µg/mL of AuNP-C in chicken embryos, subjected to oxidative damage induced by 2,2'-azobis(2-amidinopropane) dihydrochloride, significantly reduced lipid peroxidation and reactive oxygen species levels in the cardiac tissue. Moreover, the activities of cardiac superoxide dismutase, catalase, and glutathione reductase were restored, accompanied by an increase in overall antioxidant capacity. Furthermore, at higher concentrations, AuNP-C normalized the reduced glutathione content. AuNP-C preserved the normal structure of blood vessels; however, it resulted in an increase in protein carbonylation. This study provides initial evidence for the modulation of antioxidant defense mechanisms by green-synthesized AuNPs and underscores the importance of investigating the in vivo safety of phytoantioxidant-functionalized nanoparticles.

A fast and simple protocol to anaesthesia in chicken embryos.

Chicken embryos (CE) are an experimental model used as an important life science research tool worldwide, and then, adequate anesthetic protocols must be adopted to avoid the unjustifiable suffering of animals. Thus, our objective was to evaluate different anesthetic protocols in CEs using an easy inoculation route, the shell membrane (SM). We adopted the heart rate by pulse and the CE movements as a parameter of pain by assessing the vase in the chorioallantoic membrane (CAM) through the shell by a sensor of a multiparametric monitor. CEs were distributed into the following groups: (i) association of ketamine (5 mg/CE), midazolam (0.05 mg/CE) and morphine (0.15 mg/CE); (ii) ketamine (5 mg/CE) and xylazine (0.125 mg/CE); (iii) xylazine (0.0125 mg/CE) and morphine (0.15 mg/CE). The stress method used to test the anesthetic potential of the drugs was high temperature stimulation, keeping the CEs 10 cm from the fire of a Bussen nozzle for 30 s. In this experimental model, associations between different drugs decreased the pulse and the movement, indicating possible sedation. After treatment, the CE's submitted to the stress method had the heart rate and movements kept low in the groups ketamine-midazolam-morphine and ketamine-xylazine, while the non-drug-treated group increased heart rate. In a group treated with xylazine-morphine, the heart rate did not decrease, but the movement decreased after the stimulus. As the best results were the combinations of ketamine-midazolam-morphine and ketamine-xylazine, we recommend these associations for use in embryos in the final third of embryonic development in experimental protocols and euthanasia.

Lipid nanoparticles based on natural matrices with activity against multidrug resistant bacterial species.

Lately, the bacterial multidrug resistance has been a reason to public health concerning around world. The development of new pharmacology therapies against infections caused by multidrug-resistant bacteria is urgent. In this work, we developed 10 NLC formulations composed of essential oils (EO), vegetable butter and surfactant. The formulations were evaluated for long-term and thermal cycling stability studies in terms of (particle size, polydispersion index and Zeta potential). In vitro antimicrobial assays were performed using disk diffusion test and by the determination of the minimum inhibitory concentration (MIC) performed with fresh and a year-old NLC. The most promising system and its excipients were structurally characterized through experimental methodologies (FTIR-ATR, DSC and FE-SEM). Finally, this same formulation was studied through nanotoxicity assays on the chicken embryo model, analyzing different parameters, as viability and weight changes of embryos and annexes. All the developed formulations presented long-term physicochemical and thermal stability. The formulation based on cinnamon EO presented in vitro activity against strains of Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from humans and in vivo biocompatibility. Considering these promising results, such system is able to be further tested on in vivo efficacy assays.

Chicken embryos are a valuable model for the selection of Bacillus subtilis for probiotic purposes.

Bacillus subtilis (BS) has been used as an excellent probiotic; however, some BS strains seem to be opportunist pathogens or do not present inhibitory effects in the pathogenic bacteria, so the characterization of BS strains for use in animals is mandatory. This study aimed to select nonpathogenic strains of BS, which can inhibit Salmonella spp., avian pathogenic Escherichia coli (APEC), and Campylobacter jejuni (CJ) using a chicken embryo as a model. We tested nine (9) strains of BS isolated from several sources (named A to I) in in vitro by tests of mucin degradation activity, haemolytic activity, apoptosis, and necrosis in fibroblasts from chickens. After the in vitro test, we tested the remaining seven (7) strains (strains A to G) in a chicken embryo (CE) as an in vivo model and target animal. We inoculated 3 log CFU/CE of each strain via allantoic fluid at the 10th day postincubation (DPI). Each treatment group consisted of eight CEs. At the 17th DPI we checked CE mortality, gross lesions, CE weight, and whether BS strains were still viable. To perform the cytokine, total protein, albumin, and reactive C protein analysis, we collected the CE blood from the allantoic vessel and intestine fragments in the duodenum portion for histomorphometric analysis. After the results in CEs, we tested the inhibition capacity of the selected BS strains for diverse strains of Salmonella  Heidelberg (SH), S. Typhimurium (ST), S. Enteritidis (SE), S. Minnesota (SM), S. Infantis (SI), Salmonella var. monophasic (SVM), APEC and C. jejuni. After the in vitro trial (mucin degradation activity, haemolytic activity, apoptosis, and necrosis), we removed two (2) strains (H and I) that showed ß-haemolysis, mucin degradation, and/or high apoptosis and necrosis effects. Although all strains of BS were viable in CEs at the 17th DPI, we removed four (4) strains (A, B, D, F) once they led to the highest mortality in CEs or a high albumin/protein ratio. C. jejuni inoculated with strain G had greater weight than the commercial strain, which could be further used for egg inoculation with benefits to the CE. From the tests in CEs, we selected the strains C, E, and G for their ability to inhibit pathogenic strains of relevant foodborne pathogens. We found that the inhibition effect was strain dependent. In general, strains E and/or G presented better or similar results than commercial control strains in the inhibition of SH, ST, SI, APEC, and two (2) strains of CJ. In this study, we selected BS strains C, E and G due to their in vitro and in vivo safety and beneficial effects. In addition, we emphasize the value of CE as an in vivo experimental model for assessing BS's safety and possible benefits for poultry and other animals.

Physiological Changes in Chicken Embryos Inoculated with Drugs and Viruses Highlight the Need for More Standardization of this Animal Model.

Several studies have been developed using the Gallus gallus embryo as an experimental model to study the toxicity of drugs and infections. Studies that seek to standardize the evaluated parameters are needed to better understand and identify the viability of CEs as an experimental model. Therefore, we sought to verify whether macroscopic, histopathological, blood count, metabolites and/or enzymes changes and oxidative stress in CE of different ages are specific to the model. To achieve this goal, in ovo assays were performed by injecting a virus (Gammacoronavirus) and two drugs (filgrastim and dexamethasone) that cause known changes in adult animals. Although congestion and inflammatory infiltrate were visible in the case of viral infections, the white blood cell count and inflammation biomarkers did not change. Filgrastim (FG) testing did not increase granulocytes as we expected. On the other hand, CE weight and red blood cell count were lower with dexamethasone (DX), whereas white blood cell count and biomarkers varied depended on the stage of CE development. Our work reinforces the importance of standardization and correct use of the model so that the results of infection, toxicity and pharmacokinetics are reproducible.