Combined Use of NMR, LC-ESIMS and Antifungal Tests for Rapid Detection of Bioactive Lipopeptides Produced by Bacillus

The science and technology interact with the art in several ways. Biotechnological coupled with analytical approaches can play an important role in protecting and preserving cultural heritage for future generations. Many microorganisms influenced by environmental conditions are the main responsible for biological contamination in built heritage. Biocides based on chemical compounds have been used to mitigate this problem. Thus, it is vitally important to develop proper remediation actions based on environmentally innocuous alternative. Bacillus specie is emerging as an optimistic alternative for built heritage treatment due to their capacity to produce secondary metabolites with antagonistic activities against many fungal pathogens. Therefore, the intent of this work was to access a rapid evaluation of antifungal potential of bioactive metabolites produced by Bacillus strains and simultaneously their characterization using spectroscopic (NMR) and chromatographic techniques (LCESI- MS). The high antifungal activity obtained for Bacillus sp. active compounds produced in this study confirms the great potential to suppress biodeteriogenic fungi growth on historical artworks. Additionally, the proposed methodology allowed to access bioactive metabolites produced without need of the laborious total previous isolation and could be used as a viable alternative to be employed for screening and production of new green biocides.

Antioxidant,antimicrobial and toxicological properties of Schinus molle L. essential oils

Ethnopharmacological relevance: Schinus molle L. has been used in folk medicine as antibacterial, antiviral, topical antiseptic, antifungal, antioxidant, anti-inflammatory, anti-tumoural as well as antispasmodic and analgesic; however, there are few studies of pharmacological and toxicological properties of S. molle essential oils. Aim of the study: The aim of this study was to evaluate the antioxidant and antimicrobial activities of S. molle leaf and fruit essential oils, correlated with their chemical composition and evaluate their acute toxicity. Materials and methods: The chemical composition of S. molle leaf and fruit essential oils were evaluated by GC-FID and GC-MS. Antioxidant properties were determined using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical and β-carotene/linoleic acid methods. Antimicrobial properties were evaluated by the agar disc diffusion method and minimal inhibitory concentration assay. Toxicity in Artemia salina and acute toxicity with behavioural screening in mice were evaluated. Results: The dominant compounds found in leaf and fruit essential oils (EOs) were monoterpene hydrocarbons, namely -phellandrene, β-phellandrene, β-myrcene, limonene and α-pinene. EOs showed low scavenging antioxidant activity by the DPPH free radical method and a higher activity by the β-carotene/linoleic acid method. Antimicrobial activity of EOs was observed for Gram+, Gram– pathogenic bacteria and food spoilage fungi. EOs showed cytotoxicity for Artemia salina and lower toxicity in Swiss mice. Conclusions: The result showed that EOs of leaves and fruits of S. molle demonstrated antioxidant and antimicrobial properties, suggesting their potential use in food or pharmaceutical industries.

ANTIOXIDANT, ANTIMICROBIAL AND TOXICOLOGICAL PROPERTIES OF SCHINUS MOLLE L. ESSENTIAL OILS

Schinus molle L. is commonly known as pink pepper or American pepper, of Anacardiaceae family, from subtropical regions of South America, introduced and naturalized in South Europe, including Portugal. In folk medicine, plant extracts and essential oil has related as having antibacterial, antiviral, antifungal, anti-inflammatory, antitumoral, antispasmodic, analgesic and antidepressive properties. The aim of present study was to evaluate the chemical composition and biological activities of essential oil extracted from leaves and fruits of S. molle. For this purpose, the essential oils were analyzed by gas chromatography (GC/FID) and antioxidant properties were evaluated by the free radical DPPH and by system β-carotene/linoleic acid methods. The antimicrobial activities were screened against pathogenic bacteria and fungi and food spoiling fungi by the disc diffusion assay and minimal inhibitory concentration (MIC) was determined for sensitive strains. Toxicity of essential oils were carried out by the brine shrimp mortality test (EC50) and acute lethal dose (DL50) determination after oral administration in Swiss mice The major components in leaf essential oil were α-phellandrene, β-phellandrene and limonene, while myrcene, α-phellandrene and 1,8-cineole are the main components in the fruit essential oil. The essential oils of leaf and fruit of S. molle showed antioxidant activity through the two mechanisms: the ability to capture free radicals and protection of lipid peroxidation. These oils exhibited also a broad microbial activity spectrum, against pathogenic bacteria Gram-positive and Gram-negative and Candida spp. The fruit essential oil showed high cytotoxicity against Artemia salina. Essential oils of leaves and fruits of S. molle showed significant antioxidant and microbial properties, so the studies continue to clarify more in deep its toxicity, including hepatotoxicity and nephrotoxicity, and to evaluate its medicinal or nutraceutical potential.

Clínica e cirurgia em animais de companhia

Este relatório foi realizado no âmbito do estágio curricular no Hospital Veterinário do Baixo Vouga de 1 de Setembro de 2015 a 31 de Janeiro de 2016. A primeira componente trata da casuística acompanhada no estágio. A área médica mais comum foi a gastroenterologia. A segunda componente consiste na revisão bibliográfica da aspergilose canina complementada com um caso clínico acompanhado no estágio. A aspergilose sino-nasal canina ocorre principalmente em indivíduos jovens ou de meia-idade, mesaticéfalos ou dolicocéfalos e saudáveis. O seu diagnóstico implica o conjunto de vários exames, nomeadamente imagiológicos, cultura de fungos, histopatologia, serologia e diagnóstico molecular. O tratamento recomendado é o tópico. A aspergilose disseminada é menos frequente, sendo mais comum na raça Pastor Alemão. Sendo geralmente mais grave, o tratamento passa essencialmente pela terapia antifúngica sistémica. O uso de fungicidas tem sido muito associado à ocorrência de resistências cruzadas a antifúngicos azóis, dificultando o tratamento destas infeções; Abstract: Small Animal Medicine This report was elaborated following a traineeship at the Hospital Veterinário do Baixo Vouga from September 1st, 2015 to January, 31st, 2016. The first component covers the casuistry accompanied during the same. The most prevalent medical field was the gastroenterology. The second component consists of a literature review of canine aspergillosis along with the report of a case followed during the internship. Canine sinonasal aspergillosis primarily affects young to middle-aged, mesaticephalic or dolichocephalic and healthy dogs. Its diagnosis involves a conjunction of medical exams, namely imagiologic, fungal culture, histopathology, serology and molecular diagnosis. The recommended treatment is the topical one. Disseminated aspergillosis is more infrequent, occurring usually in German Shepard Dogs. Being more grievous, its treatment is based upon the administration of systemic antifungals. The use of azole fungicides has been linked to the development of cross-resistances between these and the antifungal azoles, making it difficult to treat such infections.

Green mitigation strategy for cultural heritage: bacterial potential for biocide production

Several biosurfactants with antagonistic activity are produced by a variety of microorganisms. Lipopeptides (LPPs) produced by some Bacillus strains, including surfactin, fengycin and iturin are synthesized nonribosomally by mega-peptide synthetase (NRPS) units and they are particularly relevant as antifungal agents. Characterisation, identification and evaluation of the potentials of several bacterial isolates were undertaken in order to establish the production of active lipopeptides against biodeteriogenic fungi from heritage assets. Analysis of the iturin operon revealed four open reading frames (ORFs) with the structural organisation of the peptide synthetases. Therefore, this work adopted a molecular procedure to access antifungal potential of LPP production by Bacillus strains in order to exploit the bioactive compounds synthesis as a green natural approach to be applied in biodegraded cultural heritage context. The results reveal that the bacterial strains with higher antifungal potential exhibit the same morphological and biochemical characteristics, belonging to the genera Bacillus. On the other hand, the higher iturinic genetic expression, for Bacillus sp. 3 and Bacillus sp. 4, is in accordance with the culture antifungal spectra. Accordingly, the adopted methodology combining antifungal screening and molecular data is represent a valuable tool for quick identification of iturin-producing strains, constituting an effective approach for confirming the selection of lipopeptides producer strains.

Toxicological assessment of novel green biocides for cultural heritage.

The damaging of buildings and monuments by biological contamination is a cause of serious concern. Biocides based on chemical toxic compounds have been used to mitigate this problem. However, in the past decade many of the most effective biocides have been banned due to their environmental and health hazards. Therefore, proper remediation actions for microbiologically contaminated historic materials based on environmentally safe solution is of vital importance. Bacillus species are emerging as a promising alternative for built heritage treatment. They produce a great diversity of secondary metabolites with biological activity, well known to possess antagonistic activities against many fungal pathogens. In order to evaluate the antifungal activity of the novel biocides produced in our laboratory by cultures of selected bacterial strains, liquid interaction assays using four biodeteriogenic fungi were achieved, revealing a nearly 100% of inhibitory capacity to fungal proliferation. To confirm their effective safe toxicological properties, in vivo tests using two different biological models were performed. The lyophilized supernatant of the Bacillus culture broth showed no lethality against brine shrimp and also no toxicological effects in Swiss mice through administration of acute dose of 5000 mg/kg by oral gavage. In fact, the bioactive compounds were no lethal at the tested dose unlike Preventol® (commercial biocide) that induced acute toxicity with 10 times minor concentration dose administrated in the same conditions. Therefore, the new bioactive compounds that suppress growth of biodeteriogenic fungi on historical artworks, presenting at the same time no toxicity against other living organisms, constituting an efficient and green safe solution for biodegradation/biodeterioration treatment of Cultural Heritage.