Activity of biogenic silver nanoparticles in planktonic and biofilm-associated Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a gram-positive bacterium and is the etiologic agent of caseous lymphadenitis (CL) in small ruminants. This disease is characterized by the development of encapsulated granulomas in visceral and superficial lymph nodes, and its clinical treatment is refractory to antibiotic therapy. An important virulence factor of the Corynebacterium genus is the ability to produce biofilm; however, little is known about the characteristics of the biofilm produced by C. pseudotuberculosis and its resistance to antimicrobials. Silver nanoparticles (AgNPs) are considered as promising antimicrobial agents, and are known to have several advantages, such as a broad-spectrum activity, low resistance induction potential, and antibiofilm activity. Therefore, we evaluate herein the activity of AgNPs in C. pseudotuberculosis, through the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antibiofilm activity, and visualization of AgNP-treated and AgNP-untreated biofilm through scanning electron microscopy. The AgNPs were able to completely inhibit bacterial growth and inactivate C. pseudotuberculosis at concentrations ranging from 0.08 to 0.312 mg/mL. The AgNPs reduced the formation of biofilm in reference strains and clinical isolates of C. pseudotuberculosis, with interference values greater than 80% at a concentration of 4 mg/mL, controlling the change between the planktonic and biofilm-associated forms, and preventing fixation and colonization. Scanning electron microscopy images showed a significant disruptive activity of AgNP on the consolidated biofilms. The results of this study demonstrate the potential of AgNPs as an effective therapeutic agent against CL.

Evaluation of the microbial reduction efficacy and perception of use of an ozonized water spray disinfection technology.

The development of new approaches for the decontamination of surfaces is important to deal with the processes related to exposure to contaminated surfaces. Therefore, was evaluated the efficacy of a disinfection technology using ozonized water (0.7-0.9 ppm of O3) on the surfaces of garments and accessories of volunteers, aiming to reduce the spread of microbial pathogens in the workplace and community. A Log10 microbial reduction of 1.72-2.40 was observed between the surfaces tested. The microbial reductions remained above 60% on most surfaces, and this indicated that the disinfection technology was effective in microbial log reduction regardless of the type of transport used by the volunteers and/or their respective work activities. In association with the evaluation of efficacy, the analysis of the perception of use (approval percentage of 92.45%) was fundamental to consider this technology as an alternative for use as a protective barrier, in conjunction with other preventive measures against microbiological infections, allowing us to contribute to the availability of proven effective devices against the spread of infectious agents in the environment.

Activity of Ethanolic and Supercritical Propolis Extracts in Corynebacterium pseudotuberculosis and Its Associated Biofilm.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis in small ruminants, a chronic disease characterized by the development of granulomas in superficial and visceral lymph nodes as well as in several organs. An important characteristic of the infection with this bacterium is the formation of a biofilm and the absence of effective antibiotic therapy against the disease. From this scenario, the objective of this study was to evaluate the susceptibility of C. pseudotuberculosis to conventional antibiotics and to red, green, and brown propolis extracts obtained by the supercritical and ethanolic extraction methods as well as its activity in the bacterial biofilm. The results of the sensitivity test using antibiotics indicated a sensitivity of C. pseudotuberculosis strains to the antimicrobial agents. The ethanolic extract of green propolis and the supercritical red propolis extract showed the best antibacterial activities against planktonic C. pseudotuberculosis. A lower antimicrobial activity of the brown propolis extract was identified. Propolis extracts were effective in interfering with the formation of the C. pseudotuberculosis biofilm but had little activity on the consolidated biofilm. In conclusion, propolis extracts are more effective against C. pseudotuberculosis in the planktonic stage, being able to interfere with the formation of bacterial biofilm. However, the action of propolis extracts in a sessile and structured microbial biofilm is reduced.

Ozonized Water in Microbial Control: Analysis of the Stability, In Vitro Biocidal Potential, and Cytotoxicity.

O3 dissolved in water (or ozonized water) has been considered a potent antimicrobial agent, and this study aimed to test this through microbiological and in vitro assays. The stability of O3 was accessed following modifications of the physicochemical parameters of water, such as the temperature and pH, with or without buffering. Three concentrations of O3 (0.4, 0.6, and 0.8 ppm) dissolved in water were tested against different microorganisms, and an analysis of the cytotoxic effects was also conducted using the human ear fibroblast cell line (Hfib). Under the physicochemical conditions of 4 °C and pH 5, O3 remained the most stable and concentrated compared to pH 7 and water at 25 °C. Exposure to ozonized water resulted in high mortality rates for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans. Scanning electron micrograph images indicate that the effects on osmotic stability due to cell wall lysis might be one of the killing mechanisms of ozonized water. The biocidal agent was biocompatible and presented no cytotoxic effect against Hfib cells. Therefore, due to its cytocompatibility and biocidal action, ozonized water can be considered a viable alternative for microbial control, being possible, for example, its use in disinfection processes.

Brazilian Green Propolis as a Therapeutic Agent for the Post-surgical Treatment of Caseous Lymphadenitis in Sheep.

As antibiotics are ineffective when used to treat caseous lymphadenitis, the surgical excision of lesions is often required. Iodine solution (10%) is currently the choice for the post-surgical treatment; however, it may cause histotoxicity. Propolis are resinous substances composed by a mixture of different plants parts and molecules secreted by bees. As green propolis has already proven to possess anti-bacterial and wound healing properties, this study aimed to evaluate the use of a green propolis-based ointment as a therapeutic agent for the post-surgical treatment of caseous lymphadenitis. The caseous lesions of 28 sheep were surgically excised before dividing animals into two groups: (1) iodine-treated animals and (2) sheep treated with an ointment made with a previously characterized green propolis extract. Clinical data of animals, size of the scar area, the presence of moisture and secretion in the surgical wound, the humoral immune response against the bacterium and the susceptibility of C. pseudotuberculosis clinical isolates to the green propolis extract were analyzed. The green propolis-treated group presented complete healing of the surgical wound 1 week before the iodine-treated group. Additionally, animals treated with the green propolis ointment had fewer cases of wound secretion, but it was not statistically different from the iodine-treated group. No clinical signs indicating green propolis toxicity or other side effects were found, associated with a faster and more organized hair recovery by propolis use. The green propolis extract was able to inhibit the growth of 23 from the 27 C. pseudotuberculosis clinical isolates, with minimum inhibitory and minimum bactericide concentrations ranging from 01 to 08 mg/mL, and did not interfere with the humoral immune response against the bacterium. In addition, green propolis was able to inhibit biofilm formation by four of the C. pseudotuberculosis clinical isolates. We concluded that green propolis is a promising therapeutic agent to be used in the post-surgical treatment of caseous lymphadenitis in small ruminants due to its effects on surgical wound healing, hair recovery, inhibition of wound contamination and bacterial growth.

Biogenic Silver Nanoparticles as a Post-surgical Treatment for Corynebacterium pseudotuberculosis Infection in Small Ruminants.

Caseous lymphadenitis (CL) is an infectious and zoonotic disease characterized by the development of granulomas in the lymph nodes and internal organs of small ruminants. The etiological agent of this disease is Corynebacterium pseudotuberculosis, a Gram-positive and facultative intracellular bacterium. The conventional treatment for CL consists of drainage and chemical cauterization of the lesions using a 10% iodine solution. However, this type of treatment is not effective, due to iodine's cytotoxic profile and low antibacterial activity. Currently, silver nanoparticles (AgNPs) can be seen as an alternative treatment for CL due to their antimicrobial activity and wound healing effects. Therefore, the present study aimed to evaluate AgNPs as a post-surgical treatment for CL. Twenty-nine goats and sheep with clinical signs of CL were selected. Surgical intervention was performed to excise the caseous lesions. To treat the lesions, an ointment formulation based on AgNP mixed with natural waxes and oils was used in the experimental group, and the conventional treatment with 10% iodine was used in the control group. Bacteria were isolated from the excised caseous material. The animals were observed for 8 weeks after the surgical treatment, and blood samples were taken weekly. The surgical wounds of sheep treated with AgNP healed faster, and the surgical wound area was smaller during the observation period; the latter effect was also observed in goats. AgNP-treated animals also had less purulent discharge and less moisture in the surgical wounds. The AgNP-treated animals had lower leukocyte counts and lower titers of anti-C. pseudotuberculosis antibodies. There was no statistically significant difference between the groups with regard to the hemogram results. The results of the susceptibility testing of C. pseudotuberculosis strains (T1, 1002, FRC41, and VD57 strains) and clinical isolates to AgNPs showed growth inhibition, even at low concentrations. It can be concluded that post-surgical treatment of CL using the AgNP-based ointment may be a promising tool in the control of CL, through faster healing, decreased wound contamination, and no apparent toxic effects.

Susceptibility of Malassezia pachydermatis Clinical Isolates to Allopathic Antifungals and Brazilian Red, Green, and Brown Propolis Extracts.

Clinical mycoses treatment is associated with issues such as negative side effects, high cost, prolonged treatment, and resistant strain selection. Malassezia pachydermatis is the most frequently isolated yeast in cases of canine otitis and dermatitis. The number of fungal strains exhibiting primary resistance to several drugs in vitro is increasing. Propolis has a diverse chemical composition and well-known therapeutic properties against mycoses. An alternative method for producing propolis extracts using supercritical fluid has higher selectivity, yielding extracts with fewer pollutant residues. This study therefore aimed to evaluate the in vitro susceptibility profile of M. pachydermatis clinical isolates to precharacterized supercritical and ethanolic extracts. Three types of Brazilian propolis extracts (green, red, and brown) and commercial allopathic antifungals were used in this investigation. We used the microdilution broth technique to evaluate the susceptibility profile of the yeasts. The minimum inhibitory concentration (MIC) of the brown propolis ethanolic extract was ≥16 µg/mL for all isolates. The MICs of fluconazole, ketoconazole, itraconazole, and amphotericin B ranged from 8 to >64 µg/mL, 0.032-4 µg/mL, 0.0313-16 µg/mL, and 1-2 µg/mL, respectively. The MICs of ethanolic red propolis extracts were lower than those of supercritical red propolis extracts. However, the green propolis ethanolic extract had more pronounced fungicidal activity. Isolates with lower susceptibility to commercial fungicides were inhibited by red and green propolis extracts. These results indicate that propolis can potentially be used in in vivo experiments as a promising therapeutic agent against M. pachydermatis infections.

Sensibilidade in vitro a antimicrobianos de Corynebacterium pseudotuberculosis isolados de pequenos ruminantes da região Centro- Norte do Estado da Bahia / In vitro antimicrobial susceptibility of Corynebacterium pseudotuberculosis isolated from small ruminants from the North-Central region of the State of Bahia

Introdução: corynebacterium pseudotuberculosis é uma bactéria com importância em medicina veterinária por ser o agente etiológico da Linfadenite Caseosa (LC) em pequenos ruminantes. A doença leva a perdas econômicas devido a condenação de carcaças, diminuição de produtividade e redução do valor comercial do couro dos animais acometidos. O tratamento da doença é pouco eficaz, pois os agentes antimicrobianos não conseguem atravessar a barreira dos granulomas, mas tem sido proposta a inoculação intracapsular de antibióticos como tratamento e como medida de prevenção de contaminação ambiental. Objetivo: devido a esse fato, o presente estudo teve como objetivo avaliar a sensibilidade in vitro de isolados clínicos de Corynebacterium pseudotuberculosis isolados de animais da região Centro-Norte do estado da Bahia. Metodologia: foram retirados material de granulomas de 11 animais de uma fazenda na região acima mencionada, e as bactérias foram isoladas a partir desse material. Esses isolados então foram submetidos a metodologia de difusão em disco de ágar utilizando-se discos impregnados com doze antibióticos diferentes. Resultados: 90,9% (10/11) dos isolados foram resistentes a oxacilina. Com relação à sensibilidade, 100% dos isolados foram sensíveis a ampicilina, amicacina, amoxicilina, cefalotina, ceftriaxona, ciprofloxacina, enrofloxacina, neomicina e penicilina, enquanto 90,9% destes apresentaram sensibilidade a bactericina e doxiciclina. Conclusão: pode-se concluir que os isolados de C. pseudotuberculosis da região estudada apresentam sensibilidade a diversos antibióticos, o que pode facilitar na escolha de um antimicrobiano com menor toxicidade para fins de tratamento com inoculação intracapsular do medicamento.

Background: corynebacterium pseudotuberculosisis important bacteria in veterinary medicine because it is the Etiologic Agent of Caseous Lymphadenitis (CL) in small ruminants. The disease leads to economic losses due to condemnation of carcasses, productivity decrease and leather commercial value reduction of affected animals affected. The treatment of the disease is not very effective, because antimicrobial agents cannot cross the barrier of granulomas, but it has been proposed the intracapsular inoculation of antibiotics as treatment and as a measure of prevention of environmental contamination. Objective: due to this fact, the present study aimed to evaluate the in vitro susceptibility of clinical isolates of Corynebacterium pseudotuberculosis isolated from animals in the North Central region of the State of Bahia. Methodology: granuloma material were removed from 11 animals from a farm in the above mentioned area, and the bacteria were isolated from this material. These isolated bacteria were then subjected to disk diffusion method of agar using disks impregnated with twelve different antibiotics. Results: 90,9% (10/11) the isolated bacteria were resistant to oxacillin.With respect to sensitivity, 100% of the isolates bacteria were susceptible to amikacin, ampicillin, amoxicillin, cephalotin, ceftriaxone, ciprofloxacin, enrofloxacin, neomycin and penicillin, while 90.9% of these showed sensitivity to bacitracin and doxycycline. Conclusion: it can be concluded that the isolated bacteria of C. pseudotuberculosis from the region studied present sensitivity to different antibiotics, which can facilitate the choice of a less toxic antimicrobial for treating with intracapsular inoculation of the medicine.