Transcription profiling by array of Staphylococcus aureus grown in cheese matrix in mixed culture with Lactococcus lactis
ABSTRACT: Effect of the presence of Lactococcus lactis on Staphylococcus aureus transcriptome in cheese matrix. S. aureus was co-cultured with L. lactis LD61 in cheese matrix during 7 days. RNA samples were extracted at different time points (6 h, 8 h, 10 h, 24 h and 7 days) in order to monitor the dynamic response of S. aureus MW2 in cheese matrix in presence of L. lactis
Project description:This SuperSeries is composed of the following subset Series: GSE23987: Transcriptomic profiles of six strains of Lactococcus lactis in ultrafiltration-cheese model GSE23990: Comparative genome hybridization profiles of six strains of Lactococcus lactis Refer to individual Series
Project description:The intra sub-species diversity of six strains of Lactococcus lactis subsp. lactis was investigated at the genomic level and in terms of phenotypic and transcriptomic profiles in UF-cheese model. Six strains were isolated from various sources, but all are exhibiting a dairy phenotype. Our results showed that, the six strains exhibited small phenotypic differences since similar behaviour in terms of growth was obtained during cheese ripening while only different acidification capability was detected. Even if all strains displayed high genomic similarities, sharing a high core genome of almost two thousands genes, the expression of this core genome directly in the cheese matrix revealed major strain-specific differences. This strains with the same dairy origin. Strains were cultured on skimmed raw milk ultrafiltration (UF) retentate. The UF retentate was pre-incubated overnight at 4 °C, then 45 minutes at 50 °C and homogenized during 1.5 minutes at 24 000 rpm with an ultra-turax (Imlab, France). After addition of rennet (0.3 µl ml-1), 400 g UF retentate was inoculated at 2 106 CFU/g with L. lactis subsp. lactis strains. After incubation for 8 hours at 30 °C, the cheeses were transferred at 12° C until 7 days for ripening simulation. At least three independent cultures of the six strains were performed. Total RNA was extracted from cells grown 24 hours in UF-cheese and radiolabelled cDNA were prepared and hybridized on nylon arrays. 1948 amplicons specific of Lactococcus lactis IL1403 genes were spotted twice on the array. 3 independent repetitions were performed.
Project description:The intra sub-species diversity of six strains of Lactococcus lactis subsp. lactis was investigated at the genomic level and in terms of phenotypic and transcriptomic profiles in UF-cheese model. Six strains were isolated from various sources, but all are exhibiting a dairy phenotype. Our results showed that, the six strains exhibited small phenotypic differences since similar behaviour in terms of growth was obtained during cheese ripening while only different acidification capability was detected. Even if all strains displayed high genomic similarities, sharing a high core genome of almost two thousands genes, the expression of this core genome directly in the cheese matrix revealed major strain-specific differences. This strains with the same dairy origin. Strains were cultured on M17. At least three independent cultures of the six strains were performed. Genomic DNA was extracted from cells grown overnight on M17 and radiolabelled cDNA were prepared and hybridized on nylon arrays. 1948 amplicons specific of Lactococcus lactis IL1403 genes were spotted twice on the array. 3 independent repetitions were performed.
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is problematic both in hospitals and the community. Currently, we have limited understanding of mechanisms of innate immune evasion used by S. aureus. To that end, we created an isogenic deletion mutant in strain MW2 (USA400) of the saeR/S two-component gene regulatory system and studied its role in mouse models of pathogenesis and during human neutrophil interaction. In this study, we demonstrate saeR/S plays a distinct role in S. aureus pathogenesis and is vital for virulence of MW2 in a mouse model of sepsis. Moreover, deletion of saeR/S significantly impaired survival of MW2 in human blood and after neutrophil phagocytosis. Microarray analysis of genes influenced by saeR/S demonstrated SaeR/S of MW2 influences a wide variety of genes with diverse biological functions. These data shed new insight into how virulence is regulated in S. aureus and associates a specific staphylococcal gene-regulatory system with invasive staphylococcal disease. Wild type control vs mutant at two different growth phases
Project description:The response of L. lactis to the presence of S. cerevisiae was analyzed during the exponential growth phase in fermentors in defined growth conditions. Although no growth kinetic difference was observed between the pure and mixed culture of L. lactis, the mRNA level of genes was significantly modified. More particularly, a strong reorientation of pyrimidine metabolism was observed when L. lactis was grown in the mixed culture. Keywords: microbial interaction, time course A pure culture of L. lactis was conducted in parallel with a co-culture of L. lactis and S. cerevisiae. For the transcriptomic analysis, the mixed culture (test condition, 2.5 hours of culture, B’) was compared to the pure culture (reference, 2.5 hours of culture, B) on the same slide at the same time. The mRNA level changes between 2 and 3 hours (A and C samples) flanking B sample (2.5 hours) in the pure culture were also determined on another slide. Total RNA was extracted from these samples and labelled cDNA (Cy3/Cy5) were prepared and hybridized on glass slides exhibiting 2004 amplicons specific of Lactococcus lactis IL1403 genes. 3 independent repetitions were performed.
Project description:This study aimed at establishing the effects of attenuated starters and surface bacteria on various features of caciotta cheese. The cheese undergoes a ripening period during which the house microbiota contaminates the surface. Conventional cheese (the control cheese [CC]) is made using only primary starters. Primary starters and attenuated (i.e., unable to grow and synthesize lactic acid) Lactococcus lactis (Lc. lactis) subsp. lactis were used to produce caciotta cheese without (ATT cheese) or with an inoculum of surface bacteria: (i) Leuconostoc lactis (Le. lactis) (LL cheese), (ii) Vibrio casei (VC cheese), (iii) Staphylococcus equorum (SE cheese), (iv) Brochothrix thermosphacta (BX cheese), and (v) a mixture of all four (MIX cheese). Attenuated Lc. lactis increased microbial diversity during cheese ripening. At the core, attenuated starter mainly increased indigenous lactococci and Lactobacillus delbrueckii group bacteria. At the surface, the main effect was on Macrococcus caseolyticus Autochthonous Le. lactis strains took advantage of the attenuated starter, becoming dominant. Adjunct Le. lactis positively affected Lactobacillus sakei group bacteria on the LL cheese surface. Adjunct V. casei, S. equorum, and B. thermosphacta did not become dominant. Surfaces of VC, SE, and BX cheeses mainly harbored Staphylococcus succinus Peptidase activities were higher in cheeses made with attenuated starter than in CC, which had the lowest concentration of free amino acids. Based on the enzymatic activities of adjunct Le. lactis, LL and MIX cheeses exhibited the highest glutamate dehydrogenase, cystathionine-?-lyase, and esterase activities. As shown by multivariate statistical analyses, LL and MIX cheeses showed the highest similarity for microbiological and biochemical features. LL and MIX cheeses received the highest scores for overall sensory acceptability.IMPORTANCE This study provides in-depth knowledge of the effects of attenuated starters and surface bacterial strains on the microbiota and related metabolic activities during cheese ripening. The use of attenuated Lc. lactis strongly impacted the microbiota assembly of caciotta cheese. This led to improved biochemical and sensory features compared to conventional cheese. Among surface bacterial strains, Le. lactis played a key role in the metabolic activities involved in cheese ripening. This resulted in an improvement of the sensory quality of caciotta cheese. The use of attenuated lactic acid bacteria and the surface adjunct Le. lactis could be a useful biotechnology to improve the flavor formation of caciotta cheese.
Project description:The lactococcal phage p2 is a model for studying the Skunavirus genus, the most prevalent group of phages in cheese factories worldwide. It infects L. lactis MG1363, a model strain for the study of Gram-positive bacteria. The structural proteins of phage p2 have been thoroughly described. However, most of its non-structural proteins are still uncharacterized. Here, we developed an integrative approach, making use of structural biology, genomics, physiology, and proteomics to provide insights into the function of ORF47, the most conserved non-structural protein of unknown function among the Skunavirus genus. We found this small phage protein to have a major impact on the bacterial proteome and to be important to prevent bacterial resistance to phage infection.
Project description:Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese.