Cálculo da estimativa da incerteza de medição associada ao ensaio de contagem de bactérias heterotróficas em matriz água purificada através da técnica de detecção por fluorescência / Measurement uncertainty calculation associated with heterotrophic bacterial count in purified water matrix using the fluorescence detection technique

A incerteza de medição representa o nível de confiança no resultado. Para a estimativa da incerteza de medição foi empregado o cálculo do desvio padrão da reprodutibilidade intralaboratorial de 48 ensaios de contagem de bactérias heterotróficas pela técnica da membrana filtrante com detecção por fluorescência pelo uso de substrato fluorogênico em amostras de água purificada contaminadas artificialmente entre 10 e 100 UFC/mL. O valor obtido, 1,3 x 10-3 (log10), indica que a técnica utilizada pode ser uma alternativa para a estimativa da incerteza de medição em ensaios microbiológicos quantitativos de contagem de bactérias heterotróficas em amostras de água purificada.

Measurement uncertainty represents the confidence level in the result. To estimate the expanded measurement uncertainty, the standard deviation of intra-laboratory reproducibility of 48 heterotrophic bacterial count assays by fluorescence detection by the use of fluorogenic substrate on artificially contaminated purified water samples between 10 and 100 CFU/mL was used. The value obtained, 1.3 x 10-3 (log10), indicates that the technique used can be an alternative to estimate measurement uncertainty in quantitative microbiological heterotrophic bacterial count assays in purified water samples using fluorogenic substrate.

Efeito da bactofugação nas contagens e diversidade microbiana de psicrotróficos do leite cru / Effect of bactofugation of raw milk on counts and microbial diversity of psychrotrophs

Esse estudo teve por objetivo verificar o efeito da bactofugação do leite na quantidade e diversidade de psicrotróficos. Como o leite cru era pré-aquecido (≈55ºC) para ser bactofugado, foram coletadas amostras de três lotes de leite cru refrigerado, pré-aquecido e bactofugado a 10.000g. O pré-aquecimento foi suficiente para eliminação de 99,99% dos psicrotróficos do leite cru. A bactofugação reduziu 89,66% das contagens de psicrotróficos do leite pré-aquecido. Lysinibacillus fusiformis predominou (45, 7%) no leite cru, pré -aquecido (37,5%) e bactofugado (60%). Bacillus invictae (20%), Enterococcus faecalis (10%) e Kurthia gibsonii (10%) também foram remanescentes à bactofugação, que aparentemente não tem efeito sobre uma população específica de micro-organismos, mas reduz proporcionalmente toda a microbiota psicrotrófica do leite cru.

Estudo da relação entre atividade antioxidante e contagem de bactérias láticas em iogurtes de frutas e leites fermentados comercializados em Santos-SP / Study of the relationship between antioxidant activity and bacterial count in fruit yoghurts and fermented milks available in Santos-SP

Os benefícios de probióticos e o potencial antioxidante dos alimentos estão amplamente descritos em literatura. O objetivo deste trabalho foi quantificar as bactérias lácticas presentes em produtos lácteos e analisar a atividade antioxidante desses produtos na busca por relação estatística entre esses parâmetros. Foram adquiridos produtos com alegações funcionais no rótulo e os mesmos foram submetidos à contagem de bactérias lácticas e a uma extração da atividade antioxidante, além de aferição do pH. Dentre os produtos avaliados, três deles apresentaram contagem de bactérias lácticas abaixo do esperado pela legislação vigente. Não foi possível detectar influência de variáveis na composição frente à ação antioxidante dos alimentos analisados e também não foi possível fazer associação entre a quantidade de bactérias lácticas e os valores de atividade antioxidante.

Avaliação de métodos de contagem de BAL e da qualidade microbiológica de queijo Minas Frescal comercializado no município de Duque de Caxias/RJ / Microbiological analysis of “Minas Frescal” cheese commercialized in the municipality Duque de Caxias/RJ

O queijo Minas Frescal é um derivado lácteo de amplo consumo no Brasil, entretanto suas características favorecem o crescimento de microbiota contaminante. As amostras foram submetidas às seguintes análises microbiológicas: enumeração de coliformes a 35⁰C e a 45⁰C, contagem de Staphylococcus coagulase positiva e enumeração de Bactérias Ácido Láticas (BAL). Em 10 amostras foi observada contagem de coliformes a 45⁰C acima do padrão estabelecido na legislação, confirmadas como Escherichia coli. Em todas as amostras a contagem de Staphylococcus coagulase positiva foi superior ao permitido em legislação. Nas BALs, foi observada baixa contagem em todas as amostras. Diante dos resultados obtidos, se faz necessária a adoção de medidas a fim de melhorar a qualidade microbiológica da matriz alimentícia e garantir a inocuidade alimentar.

Campylobacter in broiler slaughter samples assessed by direct count on mCCDA and Campy-Cefex agar

ABSTRACT Campylobacter spp. cause foodborne illnesses in humans primarily through the consumption of contaminated chicken. The aim of this study was to evaluate the United States Department of Agriculture's (USDA) recommended methodology, protocol MLG 41.02, for the isolation, identification and direct plate counting of Campylobacter jejuni and C. coli samples from the broiler slaughtering process. A plating method using both mCCDA and Campy-Cefex agars is recommended to recover Campylobacter cells. It is also possible to use this method in different matrices (cloacal swabs and water samples). Cloacal swabs, samples from pre-chiller and post-chiller carcasses and samples of pre-chiller, chiller and direct supply water were collected each week for four weeks from the same flock at a slaughterhouse located in an abattoir in southern Brazil. Samples were analyzed to directly count Campylobacter spp., and the results showed a high frequency of Campylobacter spp. on Campy-Cefex agar. For the isolated species, 72% were identified as Campylobacter jejuni and 38% as Campylobacter coli. It was possible to count Campylobacter jejuni and Campylobacter coli from different samples, including the water supply samples, using the two-agar method. These results suggest that slaughterhouses can use direct counting methods with both agars and different matrices as a monitoring tool to assess the presence of Campylobacter bacteria in their products.

Different methods to quantify Listeria monocytogenes biofilms cells showed different profile in their viability

Listeria monocytogenes is a foodborne pathogen able to adhere and to form biofilms in several materials commonly present in food processing plants. The aim of this study was to evaluate the resistance of Listeria monocytogenes attached to abiotic surface, after treatment with sanitizers, by culture method, microscopy and Quantitative Real Time Polymerase Chain Reaction (qPCR). Biofilms of L. monocytogenes were obtained in stainless steel coupons immersed in Brain Heart Infusion Broth, under agitation at 37 °C for 24 h. The methods selected for this study were based on plate count, microscopic count with the aid of viability dyes (CTC-DAPI), and qPCR. Results of culture method showed that peroxyacetic acid was efficient to kill sessile L. monocytogenes populations, while sodium hypochlorite was only partially effective to kill attached L. monocytogenes (p < 0.05). When, viability dyes (CTC/DAPI) combined with fluorescence microscopy and qPCR were used and lower counts were found after treatments (p < 0.05). Selective quantification of viable cells of L. monocytogenes by qPCR using EMA revelead that the pre-treatment with EMA was not appropriate since it also inhibited amplification of DNA from live cells by ca. 2 log. Thus, the use of CTC counts was the best method to count viable cells in biofilms.

Evaluation of culture media for selective enumeration of bifidobacteria and lactic acid bacteria

The purpose of this study was to test the suitability of Transgalactosylated oligosaccharides-mupirocin lithium salt (TOS-MUP) and MRS-clindamycin-ciprofloxacin (MRS-CC) agars, along with several other culture media, for selectively enumerating bifidobacteria and lactic acid bacteria (LAB) species commonly used to make fermented milks. Pure culture suspensions of a total of 13 dairy bacteria strains, belonging to eight species and five genera, were tested for growth capability under various incubation conditions. TOS-MUP agar was successfully used for the selective enumeration of both Bifidobacterium animalis subsp. lactis BB-12 and B. breve M-16 V. MRS-CC agar showed relatively good selectivity for Lactobacillus acidophilus, however, it also promoted the growth of Lb. casei strains. For this reason, MRS-CC agar can only be used as a selective medium for the enumeration of Lb. acidophilus if Lb. casei is not present in a product at levels similar to or exceeding those of Lb. acidophilus. Unlike bifidobacteria and coccus-shaped LAB, all the lactobacilli strains involved in this work were found to grow well in MRS pH 5.4 agar incubated under anaerobiosis at 37 °C for 72 h. Therefore, this method proved to be particularly suitable for the selective enumeration of Lactobacillus spp.

The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of β-galactosidase activity constitutively.Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of β-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to β-galactosidase activity .The method was found to be sensitive enough to determine 1.7 x 10(6) CFU mL-1 in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

Evaluation of the PetrifilmTM EB and TEMPO® EB systems with ISO 21528-2:2004 method for the count of Enterobacteriaceae in milk

The development of alternative microbiological techniques is driven by the necessity to meet the current needs to deliver rapid results in the manufacturing process of foods, but it is important that these methods be evaluated for each application. The objective of the present study was to assess the PetrifilmTM EB and the TEMPO® EB systems with ISO 21528-2:2004 for the count of Enterobacteriaceae in pasteurized and UHT milk samples. We analyzed the microflora of 141 pasteurized milk samples, 15 samples of artificially contaminated pasteurized milk and 15 samples of artificially contaminated UHT milk. Investigation of the method PetrifilmTM EB and ISO 21528:2 regression analysis showed a high correlation in the samples, r = 0.90 for the microflora of pasteurized milk, r = 0.98 for artificially contaminated pasteurized milk and r = 0.99 for the artificially contaminated UHT milk. In evaluating the system TEMPO EB ® method and ISO 21528:2 correlation was also significant in the analyzed samples, with r = 0.86 for the microflora of pasteurized milk, r = 0.96 for artificially contaminated pasteurized milk and r = 0.99 for artificially contaminated UHT milk. No statistically significant differences were observed between the three methods conducted to analyze artificially contaminated pasteurized and UHT milk at three inoculum levels. In conclusion, the PetrifilmTM EB system and the TEMPO® EB system may be an alternative to the ISO 21528-2:2004 for the Enterobacteriaceae assay for milk as because of the ease-of-operation and the time reduction achieved for conducting the microbiological assay using these systems.

Detection and enumeration of periodontopathogenic bacteria in subgingival biofilm of pregnant women

The aim of this study was to use the fluorescence in situ hybridization (FISH) technique to test the hypothesis of qualitative and quantitative differences of 8 periodontopathogens between pregnant and non-pregnant women. This cross-sectional study included 20 pregnant women in their second trimester of pregnancy and 20 non-pregnant women. Probing depth, bleeding on probing, clinical attachment level, and presence of calculus were recorded. Subgingival plaque samples were collected and the FISH technique identified the presence and numbers of Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia and Prevotella nigrescens. The Mann-Whitney U-test was applied to compare the data between the two groups. The mean age, ethnicity, marital status, education, and economic level in both groups were similar. The clinical parameters showed no significant differences between pregnant and non-pregnant women. The numbers of subgingival periodontopathogens were not found to be significantly different between groups, despite the higher mean counts of P. intermedia in pregnant women. Colonization patterns of the different bacteria most commonly associated with periodontal disease were not different in the subgingival plaque of pregnant and non-pregnant women.

Lower production of IL-17A and increased susceptibility to Mycobacterium bovis in mice coinfected with Strongyloides venezuelensis

The presence of intestinal helminths can down-regulate the immune response required to control mycobacterial infection. BALB/c mice infected with Mycobacterium bovis following an infection with the intestinal helminth Strongyloides venezuelensis showed reduced interleukin-17A production by lung cells and increased bacterial burden. Also, small granulomas and a high accumulation of cells expressing the inhibitory molecule CTLA-4 were observed in the lung. These data suggest that intestinal helminth infection could have a detrimental effect on the control of tuberculosis (TB) and render coinfected individuals more susceptible to the development of TB.

Diferentes métodos de isolamento de Campylobacter jejuni em alimentos / lsolation of Campylobacter Jejuni in food. Diferent methods

Esta revisão procura esclarecer a importância do Campylobacter jejuni como microrganismo emergente, incriminado em casos de infecção alimentar, quer seja em países em desenvolvimento ou desenvolvidos. Aborda ainda o perigo que as campilobacterioses podem oferecer às diferentes categorias de ingestores. Ressalta a necessidade da otimização de técnicas para detecção e isolamento de C. jejuni em produtos de origem animal e da realização de experimentos para a criação de métodos genuinamente nacionais.

The present rewiew try to clarify the importance of Campylobacter jejuni as an emergent niicrorganism which is involved in food infection, found in underdeveloped ou developed countries. This study reports also the dangerousness of campilobacteriosis to diferent corisumer's categorias. The author points out the necessity of establishing techniques to detect and isolate C. jejuni in products of animal origin and to achieve new experiments in arder to discover original national methods.