Growth of Salmonella enteritidis in artificially contaminated eggs: the effects of inoculum size and suspending media

Growth profiles of two isolates of Salmonella enteritidis phage type (PT) 4 inoculated into either the albumen of whole shell eggs or into separated albumen were found to be markedly affected by the size of the inoculum and the composition of the medium used to suspend the cells prior to inoculation. Using our model with an inoculum of two cells, multiplication of the Salmonella was not seen in 93% of eggs held at 20 degreesC for 8 days. In approximately 7% of eggs, however, growth occurred during the 8 days of storage. If the inoculum equaled or exceeded 25 cells per egg when eggs were subsequently stored at 20 degreesC, or 250 cells per egg when eggs were stored at 30 degreesC, high levels of growth of Salmonella in the egg occurred significantly more frequently than when the inoculum was two cells. High levels of growth were also seen more frequently if the inoculum was suspended in buffered peptone water or maximal recovery diluent rather than in phosphate buffered saline. Growth of Salmonella in separated albumen occurred very infrequently (1.1% of samples) at low inoculum levels and did not become significant until the inoculum was 250 cells or greater. Growth in the albumen was unaffected by the composition of the suspending medium. Provided that the inoculum was approximately 2 cells per egg and the bacteria were suspended in PBS, observed growth profiles of S. enteritidis inoculated into the albumen of whole eggs resembled those in naturally contaminated eggs. (C) 2001 Elsevier Science B.V. All rights reserved.

Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters.

Array based detection of antibiotic resistance genes in Gram negative bacteria isolated from retail poultry meat in the UK and Ireland

The use of antibiotics in birds and animals intended for human consumption within the European Union (EU) and elsewhere has been subject to regulation prohibiting the use of antimicrobials as growth promoters and the use of last resort antibiotics in an attempt to reduce the spread of multi-resistant Gram negative bacteria. Given the inexorable spread of antibiotic resistance there is an increasing need for improved monitoring of our food. Using selective media, Gram negative bacteria were isolated from retail chicken of UK-Intensively reared (n = 27), Irish-Intensively reared (n = 19) and UK-Free range (n = 30) origin and subjected to an oligonucleotide based array system for the detection of 47 clinically relevant antibiotic resistance genes (ARGs) and two integrase genes. High incidences of β-lactamase genes were noted in all sample types, acc (67%), cmy (80%), fox (55%) and tem (40%) while chloramphenicol resistant determinants were detected in bacteria from the UK poultry portions and were absent in bacteria from the Irish samples. Denaturing Gradient Gel Electrophoresis (DGGE) was used to qualitatively analyse the Gram negative population in the samples and showed the expected diversity based on band stabbing and DNA sequencing. The array system proved to be a quick method for the detection of antibiotic resistance gene (ARG) burden within a mixed Gram negative bacterial population.

Improving survival of probiotic bacteria using bacterial poly-γ-glutamic acid

A major hurdle in producing a useful probiotic food product is bacterial survival during storage and ingestion. The aim of this study was to test the effect of γ-PGA immobilisation on the survival of probiotic bacteria when stored in acidic fruit juice. Fruit juices provide an alternative means of probiotic delivery, especially to lactose intolerant individuals. In addition, the survival of γ-PGA-immobilised cells in simulated gastric juice was also assessed. Bifidobacteria strains (B. longum, B. breve), immobilised on 2.5 % γ-PGA, survived significantly better (P < 0.05) in orange and pomegranate juice for 39 and 11 days respectively, compared to free cells. However, cells survived significantly better (P < 0.05) when stored in orange juice compared to pomegranate juice. Moreover, both strains, when protected with 2.5 % γ-PGA, survived in simulated gastric juice (pH 2.0) with a marginal reduction (<0.47 log CFU/ml) or no significant reduction in viable cells after four hours, whereas free cells died within two hours. In conclusion, this research indicates that γ-PGA can be used to protect Bifidobacteria cells in fruit juice, and could also help improve the survival of cells as they pass through the harsh conditions of the gastrointestinal tract (GIT). Following our previous report on the use of γ-PGA as a cryoprotectant for probiotic bacteria, this research further suggests that γ-PGA could be used to improve probiotic survival during the various stages of preparation, storage and ingestion of probiotic cells.

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1 alpha partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B(1) and B(2) production levels. Among the isolated strains, 96 were identified as Fusarium verricillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1 alpha sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verricillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r=0.95 and r=0.79 (correlation of FUM1 with FB(1) and FB(2), respectively, P < 0.0001): r=0.93 and r =0.78 (correlation of FUM19 with FB(1) and FB(2). respectively, P < 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity. (c) 2010 Elsevier B.V. All rights reserved.

Effects of gamma radiation on the growth of Alternaria alternata and on the production of alternariol and alternariol monomethyl ether in sunflower seeds

The objective of the present study was to evaluate the effects of different gamma radiation doses on the growth of Alternaria alternata and on the production of toxins alternariol (AOH), and alternariol monomethyl ether (AME) in sunflower seed samples. After irradiation with 2, 5 and 7 kGy, the spore mass was resuspended in sterile distilled water and the suspension was inoculated into sunflower seeds. The number of colony-forming units per gram (CFU/g) was determined after culture on Dichloran Rose Bengal Chloramphenicol and Dichloran Chloramphenicol Malt Extract Agar. The presence of AOH and AME was investigated by liquid chromatography coupled to mass spectrometry. The radiation doses used resulted in a reduction of the number of A. alternata CFU/g and of AOH and AME levels when compared to the nonirradiated control group. Maximum reduction of the fungus (98.5%) and toxins (99.9%) was observed at a dose of 7 and 5 kGy, respectively. Under the present conditions, gamma radiation was found to be an alternative for the control of A. alternata and, consequently, of AOH and AME production in sunflower seeds. (C) 2009 Elsevier Ltd. All rights reserved.

Influence of package, type of apple juice and temperature on the production of patulin by Byssochlamys nivea and Byssochlamys fulva

Although the production of patulin in apple fruits is mainly by Penicillium expansum, there is no information on the ability of heat resistant moulds that may survive pasteurization to produce this mycotoxin in juice packages during storage and distribution. In this study, the production of patulin by Byssochlamys spp (Byssochlamys nivea FRR 4421, B. nivea ATCC 24008 and Byssochlamys fulva IOC 4518) in cloudy and clarified apple juices packaged in laminated paperboard packages or in polyethylene terephthalate bottles (PET) and stored at both 21 degrees C and 30 degrees C, was investigated. The three Byssochlamys strains were able to produce patulin in both cloudy and clarified apple juices. Overall, the lower the storage temperature, the lower the patulin levels and mycelium dry weight in the apple juices (p<0.05). The greatest variations in pH and degrees Brix were observed in the juices from which the greatest mycelium dry weights were recovered. The maximum levels of patulin recovered from the juices were ca. 150 mu g/kg at 21 degrees C and 220 mu g/kg at 30 degrees C. HPLC-UV, HPCL-DAD and mass spectrometry analyses confirmed the ability of B. fulva IOC 4518 to produce patulin. Due to the heat resistance of B. nivea and B. fulva and their ability to produce patulin either in PET bottles or in laminated paperboard packages, the control of contamination and the incidence of these fungi should be a matter of concern for food safety. Control measures taken by juice industries must also focus on controlling the ascospores of heat resistant moulds. (C) 2010 Elsevier B.V. All rights reserved.

Avaliação das condições higiênico-sanitárias da produção de refeições à base de carne da alimentação escolar no município de Natal-RN

The National School Nutrition Program is the oldest program in the country directed at food and nutrition safety. Its aims are to offer nutritional food as a supplement to students of public and philanthropic schools. Studying school nutrition transcends the investigation as a purely social program, given that it reaches the fields of public health, nutrition and food, using a wide variety of approaches. Thus, it is characterized by a multidisciplinary study, where the disciplines work side by side in distinct aspects of a single problem. Aim: This study aims to assess hygiene practices during the preparation of meat-based meals in public schools in the city of Natal, Brazil. Methods: A list was applied at 27 schools to identify the procedures of good food preparation practices. In addition, cooking and meal distribution temperature were measured and a microbiological analysis of the final preparation and of the water used in preparing it was performed. For microbiological analyses of the food, we analyzed coliforms at 45°C, coliforms at 35°C and Enterococcus, and for the water, we analyzed thermotolerant coliforms and total coliforms, using the methods recommended by APHA, 1995. Results: Most of the schools did not meet the required standards in all the variables related to good food preparation practices, except for the time spent preparing the meat, in which 89% were within the norm. Cooking temperature of the meals was within the standard; however, the temperature at distribution and the time spent dispensing the meals were inadequate. Of the 27 schools, 22 (81.5%) showed the presence of coliforms at 35° C in at least one meal sample and 18 (66.7%) had values above the recommended limit for coliforms at 45°C. The presence of E. coli was identified in 6.1% of the samples analyzed. The presence of Enterococcus was not found at any of the schools. With respect to the water, the North district of the city was the only one that did not meet the standards for the two indicators evaluated. The contamination found was not associated with the hygiene or food storage problems observed. Conclusions: The results show that the hygiene-sanitary conditions of meat-based public school meals were unsatisfactory, demonstrating the need for improvements in the production process to preserve the health of the student population. Multidisciplinarity: Researchers from the areas of food microbiology, nutrition, public health and statistics took part in this study, a decisive factor for characterizing the research as multidisciplinary

Avaliação das condições higiênico-sanitárias de manipuladores de alimentos e utensílios de mesa de escolas públicas municipais de Natal, RN

In Brazil school food is constitutionally guaranteed to public school students at the preschool and elementary level. This food must be nutritious, hygienic and sanitary. The aim of the present study was to assess the hygienic/sanitary conditions of food and table utensil handlers in municipal public schools in Natal, Brazil. In total, 27 public schools were assessed, using a checklist and microbiological analysis of the hands and table utensils. For the microbiological analyses of the hands, coliforms were analyzed at 45ºC and for the utensils aerobic mesophilic bacteria, using methods recognized by AOAC, 2002 and APHA, 1992, respectively. Most of the schools studied did not exhibit good food and utensil handling practice procedures in any of the variables analyzed. It was shown that 74.1% of the handlers received no periodic training, 51.9% did not undergo annual health examinations and 100% did not practice proper hand hygiene, which reflected significantly (p < 0.05) in hand contamination, where fecal coliforms were detected on 55.6% of the hands analyzed. With respect to the utensils, it was found that 100% of the schools studied did not follow correct hygiene practices and most were classified as very bad ; that is, aerobic mesophilic bacteria values above the limits established by PAHO (Pan American Health Organization), with schools in the north and south districts recording the highest percentages. The results show that the hygienic-sanitary conditions of the food and utensil handlers in the schools studied were inadequate, demonstrating the need for implanting good handling practices aimed at protecting the health of children that take part in the program and offering safer foods. Researchers from the areas of food microbiology, nutrition, public health and statistics participated in this study, a decisive factor for characterizing it as multidisciplinary

Prevalence of pigs infected by Salmonella typhimurium at slaughter after an enterocolitis outbreak

A cross-sectional study was performed to estimate the prevalence of slaughter pigs infected by Salmonella typhimurium after an enterocolitis outbreak in a commercial pig farm, which was characterised by diarrhoea during the growing phase. Anatomopathological and histopathological findings were suggestive of salmonellosis, which was further confirmed by isolation of S. typhimurium from organs and faeces samples from diseased animals. Ileocolic lymph nodes were aseptically collected from 43 pigs during slaughter procedures. The estimated prevalence of Salmonella-infected pigs was 53.48% [confidence interval (CI): 42.94:64.02%]. This finding demonstrates that the carriage of S. typhimurium at slaughter might be high if pigs originate from a batch previously affected by Salmonella-enterocolitis outbreak at the pre-harvest pork production chain. (c) 2005 Elsevier B.V. All rights reserved.

Characterization of Escherichia coli isolated from carcasses of beef cattle during their processing at an abattoir in Brazil

Beef carcass sponge samples collected between March 2003 and August 2005 at an abattoir in Brazil were surveyed for the presence of Shiga toxin-producing Escherichia coli (STEC). Only one carcass among the 80 tested showed a STEC, stx2-encoding gene by PCR amplification. The frequency of carcass contamination by E. coli during processing was tested at three situations, respectively: preevisceration, postevisceration and postprocessing, during the rain and dry seasons. The prevalence of E. coli at the three points was of 30.0%, 70.0%, 27.5% in the rain season and of 22.5%, 55.0%, 17.5% during the dry season, respectively. The E. coli isolates exhibited a high level (45.0%) of multidrug resistance to two or more antimicrobial agents. (c) 2006 Elsevier B.V. All rights reserved.

Clostridium perfringens em rações e águas fornecidos a frangos de corte em granjas avícolas do interior paulista: Brasil

Através de métodos bacteriológicos convencionais, avaliou-se a contaminação por Clostridium perfringens na ração e água utilizadas na alimentação e dessedentação de frangos de corte em diferentes regiões avícolas do interior paulista. C. perfringens esteve presente em 42 e 30% das amostras de ração e águas analisadas, respectivamente. As médias das contagens foram 6,7 x 10-2UFC mL para as amostras de água e 3,69 x 10-2UFC g para as de rações. As altas freqüências e contagens de C. perfringens verificadas nas rações e nas águas podem estar associadas à falta de higiene geral na manipulação e armazenamento dos mesmos. Sugere-se o monitoramento periódico da presença de C. perfringens nestas fontes, com a finalidade de evitar tal patógeno, em vista que o mesmo pode causar um surto de enterite necrótica levando, assim a grandes prejuízos na produção avícola.