Aplicación de la imagen hiperespectral para la detección de contaminación microbiana en espinaca fresca envasada.

La seguridad alimentaria de hortalizas de hoja puede verse comprometida por contaminaciones microbianas, lo que ha motivado la promoción mundial de normas de seguridad en la producción, el manejo poscosecha y el procesado. Sin embargo, en los últimos años las hortalizas han estado implicadas en brotes de Escherichia coli, Salmonella spp y Listeria monocytogenes, lo que obliga a plantear investigaciones que posibiliten la detección de contaminación por microorganismos con técnicas rápidas, no destructivas y precisas. La imagen hiperespectral integra espectroscopia e imagen para proporcionar información espectral y espacial de la distribución de los componentes químicos de una muestra. Los objetivos de este estudio fueron optimizar un sistema de visión hiperespectral y establecer los procedimientos de análisis multivariante de imágenes para la detección temprana de contaminación microbiana en espinacas envasadas (Spinacia oleracea). Las muestras de espinacas fueron adquiridas en un mercado local. Se sometieron a la inoculación mediante la inmersión en suspensiones con poblaciones iniciales de 5 ó 7 unidades logarítmicas de ufc de una cepa no patógena de Listeria innocua. Después de 15 minutos de inoculación por inmersión, las hojas fueron envasadas asépticamente. Se consideró un tratamiento Control-1 no inoculado, sumergiendo las espinacas en una solución tampón (agua de peptona) en lugar de en solución inoculante, y un tratamiento Control-2 en el que las hojas no se sometieron a ninguna inmersión. Las muestras se almacenaron a 8ºC y las mediciones se realizaron 0, 1, 3, 6 y 9 días después de inocular. Cada día se determinaron los recuentos microbianos y se adquirieron las imágenes con una cámara VIS-NIR (400-1000 nm). Sobre los gráficos de dispersión de los scores de PC1 y PC2 (análisis de componentes principales computados sobre las imágenes hiperespectrales considerando el rango de 500-940 nm) se reconocieron diferentes patrones que se identificaron con niveles de degradación; así se seleccionaron píxeles que conformaron las clases de no degradados, semi-degradados y degradados. Se consideraron los espectros promedio de cada clase para asignar los píxeles anónimos a una de las tres clases. Se calculó la distancia SAM (Spectral Angle Mapper) entre el espectro promedio de cada categoría y cada espectro anónimo de las imágenes. Cada píxel se asignó a la clase a la que se calcula la distancia mínima. En general, las imágenes con los porcentajes más altos de píxeles levemente degradados y degradados correspondieron a contenidos microbiológicos superiores a 5 unidades logarítmicas de ufc.

Desenvolvimento de um sumo de fruta com adição da macroalga Gelidium corneum, recorrendo à tecnologia HPP: caracterização físico-química, microbiológica e sensorial

No setor alimentar, o controlo da qualidade dos produtos e dos processos é uma etapa essencial, uma vez que é por este meio que se avaliam os padrões exigidos, quer a nível de legislação, quer a nível de mercado. O presente trabalho teve, como objetivos principais, o desenvolvimento de um novo produto alimentar com adição de algas, bem como o controlo de qualidade a nível de uma unidade fabril de produção de sumos e polpas naturais, à base de hortofrutícolas. De forma a garantir a qualidade na obtenção de produtos, foi realizado, diariamente, o controlo de entradas dos produtos hortofrutícolas, o acompanhamento de processos nas linhas de produção da fábrica e a análise das amostras de referência. Considerando a tendência e potencial de crescimento apresentados pelo setor das bebidas, nomeadamente dos sumos de fruta naturais, juntamente com a incessante procura por ingredientes naturais ricos em vários nutrientes e com propriedades bioativas, tem-se vindo a registar o lançamento de um número considerável de bebidas inovadoras, onde as macroalgas têm merecido um papel de destaque. Desta forma, procedeu-se à elaboração de dois protótipos, nomeadamente, sumo de framboesa e sumo de espinafres, com a adição do extrato da alga Gelidium corneum, estabilizados através do processo de Hiperpressão a frio, com o intuito de se avaliar o impacto desse extrato nos diferentes parâmetros de qualidade dos sumos. No que respeita aos parâmetros físico-químicos, o pH e teor de sólidos solúveis (TSS) foram avaliados após produção (t0), no tempo intermédio (t15) e no tempo final (t30). A cor foi determinada no t0 e no t30 e os restantes parâmetros, como acidez titulável, quantificação total de polifenóis, capacidade de redução do radical DPPH, teor de proteína bruta, teor de cinzas, teor de minerais e oligoelementos e teor de vitaminas (A, B1, B2 e C) foram avaliados no tempo final (t30). Foram, também, avaliados os parâmetros microbiológicos mais significativos, nomeadamente, microrganismos totais a 30 ˚C, Escherichia coli, Salmonella spp, bolores e leveduras (ao t0, t15 e t30) e realizou-se uma avaliação sensorial ao 29.º dia após produção. Estes sumos apresentaram-se microbiologicamente estáveis, não se tendo verificado crescimento microbiológico durante o período de armazenamento de 30 dias. A análise dos resultados físico-químicos permitiram constatar que a adição do extrato da alga Gelidium corneum aos sumos influenciou as suas características, tendo havido um aumento significativo do pH com a consequente diminuição da acidez titulável e um aumento do teor de sólidos solúveis, em comparação com os sumos controlo. Em relação à avaliação da cor, no caso das amostras de sumos de framboesa, verificou-se que a adição do extrato de algas influenciou o parâmetro a*. No entanto, ambas as amostras permitiram a manutenção deste parâmetro, ao longo do tempo. Quanto ao parâmetro b*, as duas amostras de sumo com algas demonstraram uma evolução mais estável, em relação aos respetivos controlos. No que respeita ao teor em minerais, a adição do extrato da alga demonstrou aumentar os teores de magnésio, sódio, potássio e iodo. Em termos sensoriais, o sumo de framboesa com algas obteve maior aceitação por parte do painel de provadores, podendo-se constatar que o atributo da cor foi o que mostrou maiores variações, entre as duas amostras.

Detecção de Escherichia coli e Salmonella spp. em microbiota intestinal de Psittaciformes em fase de reabilitação para soltura

Escherichia coli is a bacteria of the Enterobacteriacea family and it is part of the enterical microflora of mammals and of many species of birds. Salmonella spp. also belongs to the family Enterobacteriacea, it is responsible for human feed toxinfection outbreaks and usually isolated from domestic and wild birds. The present study analyzed the frequency of both agents in Psittaciformes in rehabilitation process for wildlife reintroduction. In 89 birds analyzed, 19% were infected with E. coli and 1,12% with Salmonella spp. It was carried out an analysis of the profile of antibiotic resistance in which was observed the efficiency of estreptomicin, tetraciclin, trimetoprim and gentamicin over the samples. The samples of E. coli were submitted to the Congo Red Binding test and to the Hemolisis test and 70,6% of positive samples for the first test and 53% for the second one were observed.

Detecção e identificação de genes de β-lactamases de espectro estendido em Salmonella spp. e Escherichia coli isoladas de carnes de frango, suína e bovina destinadas ao consumo humano

Pós-graduação em Microbiologia - IBILCE

Quantificação de enterobactérias e Clostridium spp. e detecção molecular de Clostridium perfringens, Escherichia coli e Salmonella spp. em pontos de cadeia produtiva da carne de frango

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The genetics and structure of the O-specific polysaccharide of Yersinia pseudotuberculosis serotype O:10 and its relationship with Escherichia coli O111 and Salmonella enterica O35

The O-specific polysaccharide (OPS) is a variable constituent of the lipopolysaccharide of Gram-negative bacteria. The polymorphic nature of OPSs within a species is usually first defined serologically, and the current serotyping scheme for Yersinia pseudotuberculosis consists of 21 O serotypes of which 15 have been characterized genetically and structurally. Here, we present the structure and DNA sequence of Y. pseudotuberculosis O:10 OPS. The O unit consists of one residue each of d-galactopyranose, N-acetyl-d-galactosamine (2-amino-2-deoxy-d-galactopyranose) and d-glucopyranose in the backbone, with two colitose (3,6-dideoxy-l-xylo-hexopyranose) side-branch residues. This structure is very similar to that shared by Escherichia coli O111 and Salmonella enterica O35. The gene cluster sequences of these serotypes, however, have only low levels of similarity to that of Y. pseudotuberculosis O:10, although there is significant conservation of gene order. Within Y. pseudotuberculosis, the O10 structure is most closely related to the O:6 and O:7 structures.

Mechanically Ventilated Broiler Sheds: a Possible Source of Aerosolized Salmonella, Campylobacter, and Escherichia coli.

This study assessed the levels of two key pathogens, Salmonella and Campylobacter, along with the indicator organism Escherichia coli in aerosols within and outside poultry sheds. The study ranged over a 3-year period on four poultry farms and consisted of six trials across the boiler production cycle of around 55 days. Weekly testing of litter and aerosols was carried out through the cycle. A key point that emerged is that the levels of airborne bacteria are linked to the levels of these bacteria in litter. This hypothesis was demonstrated by E. coli. The typical levels of E. coli in litter were similar to 10(8) CFU g(-1) and, as a consequence, were in the range of 10(2) to 10(4) CFU m(-3) in aerosols, both inside and outside the shed. The external levels were always lower than the internal levels. Salmonella was only present intermittently in litter and at lower levels (10(3) to 10(5) most probable number [MPN] g(-1)) and consequently present only intermittently and at low levels in air inside (range of 0.65 to 4.4 MPN m(-3)) and once outside (2.3 MPN m(-3)). The Salmonella serovars isolated in litter were generally also isolated from aerosols and dust, with the Salmonella serovars Chester and Sofia being the dominant serovars across these interfaces. Campylobacter was detected late in the production cycle, in litter at levels of around 107 MPN g(-1). Campylobacter was detected only once inside the shed and then at low levels of 2.2 MPN m(-3). Thus, the public health risk from these organisms in poultry environments via the aerosol pathway is minimal.

Functional transfer of Salmonella pathogenicity island 2 to Salmonella bongori and Escherichia coli.

The type III secretion system (T3SS) encoded by the Salmonella pathogenicity island 2 (SPI2) has a central role in systemic infections by Salmonella enterica and for the intracellular phenotype. Intracellular S. enterica uses the SPI2-encoded T3SS to translocate a set of effector proteins into the host cell, which modify host cell functions, enabling intracellular survival and replication of the bacteria. We sought to determine whether specific functions of the SPI2-encoded T3SS can be transferred to heterologous hosts Salmonella bongori and Escherichia coli Mutaflor, species that lack the SPI2 locus and loci encoding effector proteins. The SPI2 virulence locus was cloned and functionally expressed in S. bongori and E. coli. Here, we demonstrate that S. bongori harboring the SPI2 locus is capable of secretion of SPI2 substrate proteins under culture conditions, as well as of translocation of effector proteins under intracellular conditions. An SPI2-mediated cellular phenotype was induced by S. bongori harboring the SPI2 if the sifA locus was cotransferred. An interference with the host cell microtubule cytoskeleton, a novel SPI2-dependent phenotype, was observed in epithelial cells infected with S. bongori harboring SPI2 without additional effector genes. S. bongori harboring SPI2 showed increased intracellular persistence in a cell culture model, but SPI2 transfer was not sufficient to confer to S. bongori systemic pathogenicity in a murine model. Transfer of SPI2 to heterologous hosts offers a new tool for the study of SPI2 functions and the phenotypes of individual effectors.

Characterization of recombinant diaminopropionate ammonia-lyase from Escherichia coli and Salmonella typhimurium

Diaminopropionate ammonia-lyase gene from Escherichia coli and Salmonella typhimurium was cloned and the overexpressed enzymes were purified to homogeneity. The k(cat) Values, determined for the recombinant enzymes with DL-DAP, D-serine, and L-serine as substrates, showed that the enzyme from S. typhimurium was more active than that from E coli and the K-m values were found to be similar. The purified enzymes had an absorption maximum (lambda(max)) at 412 nm, typical of PLP dependent enzymes. A red shift in lambda(max) was observed immediately after the addition Of 10 MM DL-DAP, which returned to the original lambda(max) of 412 nm in about 4 min. This red shift might reflect the formation of an external aldimine and/or other transient intermediates of the reaction. The apoenzyme of E coli and S. typhimurium prepared by treatment With L-cysteine could be partially (60%) reconstituted by the addition of PLP. The holo, apo, and the reconstituted enzymes were shown to be present as homo dimers by size exclusion chromatography. (C) 2003 Elsevier Science (USA). All rights reserved.

Functional Analysis of the Genes Encoding Diaminopropionate Ammonia Lyase in Escherichia coli and Salmonella enterica Serovar Typhimurium

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on DL-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on DL-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on DL-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on DL-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of DL-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by DL-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by DL-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.

Reliability of Escherichia coli and faecal streptococci as indicators of Salmonella in frozen fishery products

The results obtained in the present study suggest that Escherichia coli and faecal streptococci are of little value as indicators of the possible presence of Salmonella in frozen fishery products.

Potential public health significance of faecal contamination and multidrug-resistant Escherichia coli and Salmonella serotypes in a lake in India

To assess the prevalence of faecal coliform bacteria and multiple drug resistance among Escherichia coli and Salmonella serotypes from Vembanadu Lake. Study design: Systematic microbiological testing. Methods: Monthly collection of water samples were made from ten stations on the southern and northern parts of a salt water regulator constructed in Vembanadu Lake in order to prevent incursion of seawater during certain periods of the year. Density of faecal colifrom bacteria was estimated. E. coli and Salmonella were isolated and their different serotypes were identified. Antibiotic resistance analysis of E. coli and Salmonella serotypes was done and the MAR index of individual isolates was calculated. Results: Density of faecal coliform bacteria ranged from mean MPN value 2900 -7100/100ml. Results showed multiple drug resistance pattern among the bacterial isolates. E. coli showed more than 50% resistance to amickacin, oxytetracycline, streptomycin, tetracycline and kanamycin while Salmonella showed high resistance to oxytetracycline, streptomycin, tetracycline and ampicillin. The MAR indexing of the isolates showed that they have originated from high risk source such as humans, poultry and dairy cows. Conclusions: The high density of faecal coliform bacteria and prevalence of multi drug resistant E. coli and Salmonella serotypes in the lake may pose severe public health risk through related water borne and food borne outbreaks

Survival of multi-drug resistant enteropathogenic Escherichia coli and Salmonella paratyphi in Vembanadu lake as a function of saltwater barrier along southwest coast of India

The objective of the study was to evaluate the survival response of multi-drug resistant enteropathogenic Escherichia coli and Salmonella paratyphi to the salinity fluctuations induced by a saltwater barrier constructed in Vembanadu lake, which separates the lake into a freshwater dominated southern and brackish water dominated northern part. Therefore, microcosms containing freshwater, brackish water and microcosms with different saline concentrations (5, 10, 15, 20, 25 ppt) inoculated with E. coli/S. paratyphi were monitored up to 34 days at 20 and 30 WC. E. coli and S. paratyphi exhibited significantly higher (p <0.05) survival at 20 WC compared to 30 WC in all microcosms. Despite fresh/brackish water, E. coli and S. paratyphi showed prolonged survival up to 34 days at both temperatures. They also demonstrated better survival potential at all tested saline concentrations except 25 ppt where a significantly higher (p<0.0001) decay was observed. Therefore, enhanced survival exhibited by the multi-drug resistant enteropathogenic E. coli and S. paratyphi over a wide range of salinity levels suggest that they are able to remain viable for a very long time at higher densities in all seasons of the year in Vembanadu lake irrespective of saline concentrations, and may pose potential public health risks during recreational activities

The incidence ,Antibiotic resistence and survival of salmonella and escherichia coli isolated from broiler chicken retail outlets

The incidence of salmonella and escherichia coli in chicken retail outlets in a residential area of coimbatore, Tamilnadu India was studied with the view that accessories may be a source of cross contamination.Accessories like cages,knives ,chopping boards weighing balance trays and the hands of butcher were examined.A toatal of 14 salmonella as well as 31 E.coli were isolated from different sources. The incidence of E.coli was higher than that of Salmonella.The highest incidence of Salmonella was found in chopping boards and the maximum level of E.Coli was detected in cages.

A survey of fluoroquinolone resistance in Escherichia coli and thermophilic Campylobacter spp. on poultry and pig farms in Great Britain

Aims: To estimate the proportions of farms on which broilers, turkeys and pigs were shedding fluoroquinolone (FQ)-resistant Escherichia coli or Campylobacter spp. near to slaughter. Methods and Results: Freshly voided faeces were collected on 89 poultry and 108 pig farms and cultured with media containing 1.0 mg l(-1) ciprofloxacin. Studies demonstrated the specificity of this sensitive method, and both poultry and pig sampling yielded FQ-resistant E. coli on 60% of farms. FQ-resistant Campylobacter spp. were found on around 22% of poultry and 75% of pig farms. The majority of resistant isolates of Campylobacter (89%) and E. coli (96%) tested had minimum inhibitory concentrations for ciprofloxacin of >= 8 mg l(-1). The proportion of resistant E. coli and Campylobacter organisms within samples varied widely. Conclusions: FQ resistance is commonly present among two enteric bacterial genera prevalent on pig and poultry farms, although the low proportion of resistant organisms in many cases requires a sensitive detection technique. Significance and Impact of the Study: FQ-resistant bacteria with zoonotic potential appear to be present on a high proportion of UK pig and poultry farms. The risk this poses to consumers relative to other causes of FQ-resistant human infections remains to be clarified.