RESUMEN
Transcriptional approaches are increasingly used to compare the behaviour of pathogenic and non-pathogenic bacteria in different culture conditions. The purpose of this study was to apply these methods in cheese to better characterize food and clinical Enterococcus faecalis isolates during cheese processing. Because of the complex biochemical composition of the cheese matrix, e.g. the presence of casein and fat, we developed an efficient method to recover total RNA from bacteria in a semi-hard cheese model. To validate the RNA extraction method, we analysed expression of 7 genes from two E. faecalis strains (one clinical and one food isolate) in both cheese and culture medium by semi-quantitative RT-PCR. We then used PCR-based DNA macro-arrays to compare expression of 154 genes from two E. faecalis strains in both cheese and culture medium. The food strain isolated from cheese is transcriptionally active in cheese, as reflected by the higher transcript levels of various genes. Conversely, overall transcript levels of the V583 clinical isolate were lower in cheese, suggesting that the food strain may be more adapted to a dairy environment than the clinical strain. The method described here constitutes a very promising tool for future transcriptomic studies in cheese matrices. Global profiling in foods may prove to be a valid criterion for differentiating food from clinical isolates.
Asunto(s)
Proteínas Bacterianas/genética , Queso/microbiología , Enterococcus faecalis/aislamiento & purificación , Microbiología de Alimentos , Expresión Génica , Infecciones por Bacterias Grampositivas/microbiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas Bacterianas/metabolismo , Enterococcus faecalis/genética , Humanos , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificaciónRESUMEN
Although Leuconostoc genus is "generally recognized as safe" (GRAS), a few clinically human infections cases by this microorganism have been reported in the literature, leading to their classification as opportunistic pathogens. However, these reported cases concern only severe immunodepressed patients, and none direct relations have yet been proven between Leuconostoc isolation and human diseases. Moreover, no cases of infections have been directly linked to the consumption of fermented food. Considering the long history of use of Leuconostoc in dairy industry, and their poor incidence in human infections cases, this bacterial genus may be reasonably considered as " safe " for its use in fermented dairy products.
Asunto(s)
Seguridad de Productos para el Consumidor , Productos Lácteos Cultivados/microbiología , Leuconostoc/clasificación , Filogenia , Medición de Riesgo , Microbiología de Alimentos , Humanos , Huésped Inmunocomprometido , Leuconostoc/aislamiento & purificación , Leuconostoc/patogenicidad , Especificidad de la EspecieRESUMEN
The aim of this work was to identify the bacterial biodiversity of traditional Zabady fermented milk using PCR-temporal temperature gel electrophoresis (PCR-TTGE) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Most of the identified bacterial species in Zabady samples belonged to lactic acid bacteria (LAB), e.g., Streptococcus thermophilus, Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis, Leuconostoc citreum, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus johnsonii. Using the culture-dependent and independent methods, the streptococcal and lactococcal groups appeared to be the major bacterial species in Zabady fermented milk, whereas the lactobacilli were the minor ones. The main dominant species was St. thermophilus followed by Lc. garvieae. Other molecular tools, e.g., species-specific PCR assay and cloning and sequencing strategy were used to confirm the TTGE and DGGE results. Lc. garvieae, Lc. raffinolactis, Ln. citreum, and Lb. johnsonii were identified for the first time in this type of Egyptian fermented milk.
Asunto(s)
Productos Lácteos Cultivados/microbiología , ADN Bacteriano/análisis , Lactococcus/aislamiento & purificación , Filogenia , Streptococcus/aislamiento & purificación , Biodiversidad , Recuento de Colonia Microbiana , Egipto , Electroforesis en Gel de Campo Pulsado/métodos , Microbiología de Alimentos , Lactobacillus/clasificación , Lactobacillus/aislamiento & purificación , Lactococcus/clasificación , Leuconostoc/clasificación , Leuconostoc/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Especificidad de la Especie , Streptococcus/clasificaciónRESUMEN
Ragusano cheese is a "protected denomination of origin" cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called "tinas" were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii, and Enterococcus faecium. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected, S. thermophilus being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (S. thermophilus, L. lactis, and L. delbrueckii subsp. lactis) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system.
Asunto(s)
Bacterias/clasificación , Bacterias/metabolismo , Biopelículas/crecimiento & desarrollo , Queso/microbiología , Leche/microbiología , Animales , Bacterias/crecimiento & desarrollo , Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , Electroforesis en Gel de Poliacrilamida/métodos , Fermentación , Microbiología de Alimentos , Temperatura , MaderaRESUMEN
AIMS: Fungi could be responsible for several problems in wines but the fungal ecosystem of grapes remains little known. The use of traditional methods does not allow to describe quickly this ecosystem. Therefore, we need to improve the knowledge about these fungi to prevent defects in wine. This study aims at evaluating the potentialities of the temporal temperature gradient gel electrophoresis (TTGE) method to describe the fungal ecosystem of grapes. METHODS AND RESULTS: The internal transcribed spacer (ITS) region was amplified and analysed using TTGE. A reference database of 56 fungal species was set up to evaluate the discrimination power of the method. The database was used for the direct identification of the fungal species present in complex samples. The sensitivity of the method is below 10(4) spores per ml. CONCLUSIONS: This method allows to describe the fungal diversity of grapes, but does not always allow to directly identify all fungal species, because of the taxonomic resolution of the ITS sequences. However, this identification strategy is less time consuming than traditional analysis by cloning and sequencing the bands. SIGNIFICANCE AND IMPACT OF THE STUDY: With this method, it will be possible to compare the fungal species present in different vineyards and to connect the presence of some fungi with particular defects in wine.
Asunto(s)
Hongos/genética , Microbiología Industrial , Vitis/microbiología , ADN de Hongos/análisis , Bases de Datos Genéticas , Electroforesis en Gel de Poliacrilamida , Variación Genética , EsporasRESUMEN
Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of approximately 150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.