Roberta Hilsdorf Piccoli

Formation of biofilms by Staphylococcus aureus on stainless steel and glass surfaces and its resistance to some selected chemical sanitizers

The objectives of this work were to verify the capability of Staphylococcus aureus of forming bio-film on stainless steel and glass surfaces; to evaluate the efficiency of sodium dichloroisocyanurate, hydrogen peroxide and peracetic acid in inactivating Staphylococcus aureus cells adhered onto these surfaces; and to visualize biofilm development by scanning electron microscopy before and after sanitizer treatment. The surfaces studied consisted of 10x20mm chips immersed in Petri dishes containing BHI broth inoculated with S. aureus ATCC 25923. Biofilm formation was observed after 15-day incubation, when the cells were removed using the swab technique, followed by Baird Parker agar plating. Also, the efficiency of the chemical sanitizers on the chip surfaces was tested and the non-removed cells were counted on the Baird-Parker agar. After biofilm formation and use of sanitizers, the chips were respectively observed by scanning electronic microscopy following a pre-existing protocol. The obtained results showed biofilm formation on both surfaces, with bacterial count in the order of 107 CFU/cm2 on and 108 CFU/cm2 on stainless steel and glass surfaces, respectively. Peracetic acid was the most efficient in removing adhered cells, presenting 5.26 and 4.5 decimal reduction for adhered cells on stainless steel and glass surfaces, respectively.

Antimicrobial activity of Satureja montana L. essential oil against Clostridium perfringens type A inoculated in mortadella-type sausages formulated with different levels of sodium nitrite

This research evaluated the antimicrobial effect of the winter savory (Satureja montana L.) essential oil (EO) against Clostridium perfringens type A (ATCC 3624) inoculated in mortadella-type sausages formulated with different levels of sodium nitrite (NaNO₂: 0 ppm, 100 ppm and 200 ppm) in addition to EO at concentrations of 0.0%, 0.78%, 1.56% and 3.125% stored at 25°C for 30 days. The EO extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (CG-MS) was tested in vitro using an agar well diffusion method for determination of minimum inhibitory concentration (MIC) on C. perfringens. According to compositional analysis of the winter savory EO, 26 chemical compounds were identified, and the major constituents were thymol (28.99%), p-cymene (12.00%), linalool (11.00%) and carvacrol (10.71%). The results obtained showed that EO applied at a concentration of 1.56%, which was defined as the MIC, exhibited antimicrobial activity against C. perfringens in the in vitro assays, and the transmission electron microscopy (TEM) revealed structural damage and cell lysis of C. perfringens caused by EO treatment. A synergistic effect between NaNO₂ and EO was observed. In mortadella-type sausages formulated with 100 ppm of NaNO₂ and EO at all concentrations tested, the population of target microorganisms was reduced (p≤0.05) compared to control samples during all storage period. This data suggests the potential combined use of savory EO and minimal amounts of the synthetic additive, NaNO₂ to control C. perfringens in mortadella, which goes according to current market trends, where consumers are requesting natural products.

Biofilm formation by Listeria monocytogenes on stainless steel surface and biotransfer potential.

An experimental model was proposed to study biofilm formation by Listeria monocytogenes ATCC 19117 on AISI 304 (#4) stainless steel surface and biotransfer potential during this process. In this model, biofilm formation was conducted on the surface of stainless steel coupons, set on a stainless steel base with 4 divisions, each one supporting 21 coupons. Trypic Soy Broth was used as bacterial growth substrate, with incubation at 37 °C and stirring of 50 rpm. The number of adhered cells was determined after 3, 48, 96, 144, 192 and 240 hours of biofilm formation and biotransfer potential from 96 hours. Stainless steel coupons were submitted to Scanning Electron Microscopy (SEM) after 3, 144 and 240 hours. Based on the number of adhered cells and SEM, it was observed that L. monocytogenes adhered rapidly to the stainless steel surface, with mature biofilm being formed after 240 hours. The biotransfer potential of bacterium to substrate occurred at all the stages analyzed. The rapid capacity of adhesion to surface, combined with biotransfer potential throughout the biofilm formation stages, make L. monocytogenes a potential risk to the food industry. Both the experimental model developed and the methodology used were efficient in the study of biofilm formation by L. monocytogenes on stainless steel surface and biotransfer potential.

A Weibull model to describe antimicrobial kinetics of oregano and lemongrass essential oils against Salmonella Enteritidis in ground beef during refrigerated storage.

The antimicrobial effect of oregano (Origanum vulgare L.) and lemongrass (Cymbopogon citratus (DC.) Stapf.) essential oils (EOs) against Salmonella enterica serotype Enteritidis in in vitro experiments, and inoculated in ground bovine meat during refrigerated storage (4±2 °C) for 6 days was evaluated. The Weibull model was tested to fit survival/inactivation bacterial curves (estimating of p and δ parameters). The minimum inhibitory concentration (MIC) value for both EOs on S. Enteritidis was 3.90 μl/ml. The EO concentrations applied in the ground beef were 3.90, 7.80 and 15.60 μl/g, based on MIC levels and possible activity reduction by food constituents. Both evaluated EOs in all tested levels, showed antimicrobial effects, with microbial populations reducing (p≤0.05) along time storage. Evaluating fit-quality parameters (RSS and RSE) Weibull models are able to describe the inactivation curves of EOs against S. Enteritidis. The application of EOs in processed meats can be used to control pathogens during refrigerated shelf-life.

Reduction of Aeromonas hidrophyla biofilm on stainless stell surface by essential oils

This study demonstrates the possibility of using sanitizing detergents based on natural products for the elimination and/or reduction of Aeromonas hydrophila biofilm formed on stainless steel surfaces. The goal of this work was to determine the reduction effect of sanitizing detergents containing essential oils of Thymus vulgaris (thyme) and Cymbopogon citratus (lemongrass) on biofilm formed by A. hydrophila on AISI 304 stainless steel coupons, using UHT skimmed milk as substratum. There was adhesion and biofilm formation by A. hydrophila at 28 °C, presenting 7.60 log cfu.cm−2 after the fourth day of cultivation. There was no significant difference between the lemongrass treatment and that of the thyme oil (p < 0.05). However, both treatments significantly reduced the biofilm, differing significantly from the NaOH control (p > 0.05). The treatment with lemongrass solution reduced the biofilm by 4.51 log cfu cm−2 at 25 °C. The thyme detergent also reduced the number of cfu cm−2 by 3.84 log cycles at 25 °C. The use of the lemongrass and thyme solutions efficiently reduced the A. hydrophila biofilm.

Avaliação higiênico-sanitária de linguiças tipo frescal comercializadas nos municípios de Três Corações e Lavras MG

Com o presente trabalho, objetivou-se avaliar a qualidade higienico-sanitaria de linguicas tipo frescal, em funcao do potencial risco que estes produtos representam para a saude publica. Para a conducao de tal pesquisa, amostras foram aleatoriamente coletadas no municipio de Tres Coracoes (n=20) e Lavras (n=20), ambos situados no Estado de Minas Gerais. As analises microbiologicas, conduzidas de acordo com o ICMSF (1982), constaram da enumeracao de coliformes totais e termotolerantes (colimetria), quantificacao e identificacao bioquimica de Staphylococcus coagulase positiva e Salmonella spp. Nao foi detectada a presenca de Salmonella spp. em nenhuma das amostras. Os valores para contaminacao por coliformes termotolerantes encontraram-se dentro do intervalo de 101 a 104 NMP/g. Em 14% das 40 amostras analisadas foi detectado Staphylococcus coagulase positiva acima do limite estabelecido pela legislacao vigente. Pelos valores encontrados, as linguicas analisadas nao sao produzidas dentro de padroes de higiene satisfatorios, sendo um potencial causador de toxinoses e infeccoes alimentares aos consumidores.

Formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo

The aim of this research was to verify the capability of biofilm formation on stainless steel by Aeromonas hydrophila and Staphylococcus aureus using milk and different conditions of cultivation. The variables consisted in mono and multi-species cultivation of these microorganisms and in the temperatures of 4, 7 and 18 °C. Containers containing 1000 mL of milk, population density of 10 5 CFU.mL –1 of each microorganism, and ten suspended chips of stainless steel AISI 304 (10 × 20 mm) were used to seal up and storage, under 60 rpm of agitation for 10 days. The analyses were conducted every 48 hours. Sessile cells of A. hydrophila and S. aureus and were enumerated by plating in m-Aeromonas selective agar and Baird-Parker agar. Complementary studies included the generation time calculation, enumeration of planktonic cells, and visualization of chips by scanning electron microscopy. S. aureus, in mono-cultivation, formed biofilm at 18 °C and at 7 °C. At 4 °C was observed attachment. The presence of A. hydrophila reduced the performance of S. aureus. In this condition, S. aureus formed biofilm at 18 °C. A. hydrophila formed biofilm under all conditions.

Rendimento, composição química e atividade antilisterial de óleos essenciais de espécies de Cymbopogon

RESUMO: Objetivou-se determinar o rendimento, a composicao quimica e avaliar a atividade antilisterial dos oleos essenciais de Cymbopogon citratus (DC.) Stapf. (capim-limao) e Cymbopogon nardus (L.) Rendle (capim-citronela), puros e em combinacao. A obtencao dos oleos essenciais foi realizada a partir de folhas frescas, empregando-se a tecnica de hidrodestilacao e utilizandose aparelho de Clevenger modificado. Os rendimentos foram calculados a partir da massa obtida na extracao e da umidade do material vegetal. A analise qualitativa foi realizada por cromatografia em fase gasosa acoplada a espectrometria de massas e a avaliacao dos teores dos constituintes quimicos por cromatografia em fase gasosa com detector de ionizacao de chamas. O efeito bacteriostatico de diferentes concentracoes dos oleos essenciais, puros e em combinacao, foi determinado pela tecnica de difusao em agar. As concentracoes minimas inibitorias (CMIs) foram utilizadas na determinacao do tempo de acao bactericida. O rendimento, em base seca, foi de 1,39% (v/p) para o oleo essencial de C. citratus e de 2,27% (v/p) para o oleo essencial de C. nardus. Geranial e neral foram os constituintes majoritarios do oleo essencial de C. citratus, enquanto, para C. nardus, foram citronelal, geraniol e citronelol. A maioria das concentracoes utilizadas mostrou-se efetiva contra Listeria monocytogenes e observaram-se efeitos bactericidas nas CMIs. Oleos essenciais de C. citratus e C. nardus, puros e em combinacao, constituem novas alternativas de antibacterianos naturais a serem utilizados no controle de L. monocytogenes na industria de alimentos. Palavras-chave: Cymbopogon citratus, Cymbopogon nardus, oleos essenciais, atividade antibacteriana, Listeria monocytogenes

Qualidade de produtos minimamente processados e comercializados em gôndolas de supermercados nas cidades de Lavras - MG, Brasília - DF e São Paulo - SP

A producao de frutos e hortalicas minimamente processados mostrou crescimento relevante nos ultimos anos, em razao de acentuadas mudancas no estilo de vida do consumidor, como reducao das familias, busca de conveniencia e conscientizacao da necessidade de uma dieta alimentar saudavel e que atenda as exigencias de seguranca alimentar. Objetivou-se, neste trabalho, caracterizar a qualidade quimica, fisico-quimica e microbiologica dos produtos minimamente processados, coletados em gondolas de supermercados em Lavras - MG, Brasilia - DF e Sao Paulo - SP para orientar seus fornecedores e consumidores quanto a necessidade de maior controle de qualidade, devido aos riscos de contaminacao de materias-primas por microrganismos patogenicos e deteriorantes. 144 amostras foram coletadas e submetidas as determinacoes de pH, acidez titulavel e solidos soluveis e avaliacoes microbiologicas (coliformes a 45oC, Escherichia coli e Salmonella sp.). Os resultados mostraram variacoes significativas nos valores de pH e foi observada diminuicao de acidez e solidos soluveis, durante o armazenamento. Foi detectada contaminacao por coliformes a 45oC sendo que, em 50% dessa presenca, houve isolamentos de Escherichia coli, evidenciando contaminacao oriunda de materia-prima inadequadamente higienizada ou por sua presenca nos manipuladores. Contudo, nao foi detectada contaminacao por Salmonella sp.. Os resultados obtidos indicaram a necessidade de implementacao de Boas Praticas de Fabricacao (BPF), no controle de qualidade desses produtos.

Identificação e resistência a antimicrobianos de espécies de Aeromonas móveis isoladas de peixes e ambientes aquáticos

Com o objetivo de se verificar a diversidade de especies de Aeromonas moveis e seu perfil de susceptibilidade a antimicrobianos em pisciculturas comerciais, foram selecionadas oito tilapiculturas localizadas na regiao do Alto Rio Grande, Minas Gerais. De cada propriedade foram coletadas tres amostras de peixes em estadio de pre-abate (vivos e saudaveis), uma amostra de agua do tanque e uma amostra da agua de abastecimento do sistema. De cada peixe foram coletadas amostras de lavado superficial e do parenquima renal. Diluicoes seriadas adequadas de cada amostra foram plaqueadas em TSA-ampicilina (10 mg/l) e as amostras de rim em Agar Sangue de cavalo a 5%. A partir de colonias isoladas positivas para o teste da oxidase foram realizados testes para identificacao do genero (testes presuntivos) e das especies de Aeromonas (testes bioquimicos). O perfil de antibiograma foi obtido pelo teste de difusao de discos de antibioticos em Agar Mueller Hinton. Foram obtidos 75 isolados diferenciados em nove especies de Aeromonas: A. jandaei, A. hydrophila, A. trota, A. caviae, A. sobria, A. eucrenophila, A. veronii bt veronii, A. schubertii, A. media, alem de amostras classificadas como Aeromonas atipicas. Do total isolado, oito amostras foram provenientes da superficie corporea de peixes, 14 da agua de abastecimento e 53 da agua do tanque. Nao houve isolamento a partir dos especimes de parenquima renal. Em relacao ao perfil de resistencia, 93% dos isolados foram resistentes a eritromicina, 36% a tetraciclina, 13% ao ac. nalidixico, 9% a gentamicina, 8% a nitrofurantoina, 8% a canamicina, 5% a norfloxacina, 4% ao cloranfenicol e 3% as sulfonamidas. Dentre os isolados analisados, 43% apresentaram indice de multipla resistencia a antimicrobianos (MAR) igual ou superior a 22%, ou seja, resistencia a dois ou mais drogas das nove testadas. Os dados apontam para um risco iminente, tanto pelo isolamento de amostras potencialmente patogenicas a seres humanos quanto pelo perfil de multirresistencia dos isolados.