Single-Cell Growth Probability of Listeria monocytogenes at Suboptimal Temperature, pH, and Water Activity

The single-cell growth probability of Listeria monocytogenes was characterized in tryptone soy broth supplemented with 0.6% yeast extract at temperature ranging from 5 to 25°C, pH ranging from 4.4 to 6.5, and water activity ranging from 0.919 to 0.989. Growth was monitored visually in 96-microwell plates and the growth probability was deduced from concentrations estimated with the MPN calculation. Models were proposed to describe the increase of the probability from 0 to 1 with increasing values of environmental factors. An exponential model was used for the temperature and the individual minimal temperature for growth ranged from −3.6°C [−4.5°C, −2.7°C] to 17.3°C [16.0°C, 18.6°C]. An inverse exponential model was convenient for the pH and the minimal pH for growth of individual cells ranged from 4.34 [3.93, 4.75] to 5.93 [4.85, 7.01]. A linear model was used for the water activity and the minimal value for growth of individual cells ranged from 0.917 [0.909, 0.925] to 0.988 [0.966, 1]. In spite of the great between-experiment variability, the minimal values estimated for the growth limits of individual cells were in accordance with values usually proposed for L. monocytogenes populations. This study provides models allowing the prediction of the growth probability of a few cells contaminating chilled foods with suboptimal pH and water activity improving thus the assessment of the behavior of L. monocytogenes cells naturally contaminating foods

Identification of Metabolic Pathways Expressed by Pichia anomala KH6 in the Presence of the Pathogen Botrytis cinerea on Apple: New Possible Targets for Biocontrol Improvement

peer reviewe

Evaluation of two protein extraction protocols for Pichia anomala proteome analysis.

Pichia anomala (strain Kh6) was isolated from the surface of apple fruits and selected for its high and reliable biocontrol activity against Botrytis cinerea and Penicillium expansum. Its main modes of action have until now been studied using essentially microbiological and molecular approaches. The study continues now using the proteomic approach and considering the in situ P. anomala/B. cinerea/apple interaction. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of the most powerful tools used for proteomic analysis. It combines two sequential separation steps, the first dimension via isoelectric focusing (IEF) and the second one by SDS PAGE. Although recent advances in 2-D PAGE, the extraction of the whole proteome and the removal of interfering contaminants still limit its application. Sample preparation constitutes indeed a critical influential step for IEF which in turn affects 2-D gel quality. The objective of the present work was thus to develop an effective protein extraction protocol designed for 2-D PAGE analysis of the proteome of P. anomala strain Kh6. As a starting point, two contrasting protein extraction protocols were chosen to be evaluated in terms of protein yield and one-dimensional (1-D) SDS PAGE and 2-D PAGE gel patterns. The first protocol uses a urea/thiourea-based lysis buffer whereas the second protocol utilizes a hot SDS-based lysis buffer with an additional precipitation step. The comparison model used consisted of apples treated with strain Kh6 alone (K) and apples first treated with Kh6 and then inoculated with B. cinerea conidia (KB). Growth kinetics of strain Kh6 on wounded apples was determined and found to be not affected by the presence of B. cinerea conidia. Proteins were extracted from yeast pellets collected at both the exponential and stationary phases of strain Kh6 growth on apples. The evaluation of both extraction protocols indicates that more proteins were extracted with the SDS protocol and, according to 1-D assays, higher molecular weight proteins were obtained with the ‘urea/thiourea’ protocol and, regardless of the protocol used, more bands were obtained during the exponential phase. 2-D assays are currently underway and the corresponding results will be presented

The diagnostic efficacy and safety of stress-only supine and prone myocardial perfusion imaging with a dedicated cardiac gamma camera in patients with suspected or known coronary artery disease

BACKGROUND: Myocardial perfusion scintigraphy remains one of the substantial noninvasive diagnostic methods in coronary artery disease. Recent technological advancement allowed to create novel semiconductor, dedicated cardiac gamma camera with better spatial resolution and higher energy resolution, resulting in the reduction of radiation burden and acquisition time. The aim of this study was to evaluate the efficacy and safety of stress-only supine and prone MPS with a cardiac gamma camera in patients with suspected or known coronary artery disease. MATERIAL AND METHODS: A total number of 203 consecutive patients with suspected or known coronary artery disease, who underwent MPS were enrolled in the study. The patients without perfusion abnormalities on stress supine and prone MPS scans had no rest MPS, in the remaining patients two-day stress-rest imaging was performed. The group of 160 patients with one-year follow up was subjected to final analysis. RESULTS: Stress-only protocol of myocardial perfusion imaging was performed in 72 patients, 88 patients underwent two-day stress and rest myocardial perfusion scintigraphy. In 46 out of 72 stress-only group of patients, prone study did not affect further proceedings. However, in over 1/3 of cases (26/72), prone scans resulted in abstaining from rest imaging. One year follow-up revealed no sudden cardiac deaths or myocardial infarctions in both (stress-only and stress-rest) groups. Revascularization was performed most often in the double-positive group — patients with significant ischaemia on myocardial perfusion images and chest pain or electrocardiographic changes or both during the stress test. In this double-positive group, all 11 patients had coronary angiography (two of them prior to myocardial perfusion scintigraphy), nine of them had subsequent revascularization. CONCLUSIONS: In patients with no significant perfusion abnormalities on stress scans omitting rest study is safe with very good one-year risk prognosis of acute cardiac events and allows to limit the radiation exposure and procedure duration. Additional prone acquisitions are valuable supplements in determining the decision of safe early completion of myocardial perfusion imaging

Biocontrol proteomics:Implication of the pentoses phosphates pathway in the antagonist effect of Pichia anomala against Botrytis cinerea on apple

The growing interest of the consumers for the wholesome food and the protection of the environment as well as the development of resistant pathogens to pesticides, stimulate the interest of growers to apply biological control methods. Pichia anomala strain K was previously identified as an efficient biocontrol agent of the main apple pathogens, Botrytis cinerea and Penicillum expansum. Further study demonstrated the complexicity of the mode of action of P. anomala against B. cinerea. A cDNA-AFLP and gene disruption study revealed implication of exo-β-1,3-glucanases in the mode of action of P. anomala strain Kh6 (a haploid form of P. anomala strain K displaying the same biocontrol properties). However, these studies suggested also implication of other factors. The present study aims to increase our knowledge of the mode of action of P. anomala strain Kh6 against B. cinerea using an in situ approach allowing the triple interaction, host/pathogen/antagonist and the proteomic tool allowing to study the ultime expression of the genome without a priori. One 50mm wound per apple were covered by a membrane and inoculated by a P. anomala suspension then by B. cinerea or not. Samples were collected during the exponential and stationary phase to identify the early and later responses to the presence of B. cinerea. After extraction, proteins were separated on 2-D gels. Spots influenced by the presence of B. cinerea in exponential and stationary phases were identified by MALDI-ToF. One hundred five and sixty spots of proteins were influenced by the presence of B. cinerea in exponential and stationary phases respectively. In exponential phase, P. anomala Kh6 in absence of B. cinerea uses mainly the glycolysis pathway, whereas in presence of pathogen, it orientates its energetic metabolism to the oxidative phosphorylation and sets up the pentose phosphate pathway. Thanks to this new orientation, P. anomala Kh6 probably obtains energy and nucleic acids allowing to colonize the wound as fast as in absence of B. cinerea and prevents the use of nutrients by the pathogen. In stationary phase, no differences in the P. anomala Kh6 energetic metabolism, in absence and in presence of B. cinerea were observed. During that phase, P. anomala Kh6 seems to use the alcoholic fermentation in order to face the nutrients impoverishment of the substrate

Genome sequence of the necrotrophic plant pathogen Alternaria brassicicola Abra43

Alternaria brassicicola causes dark spot (or black spot) disease, which is one of the most common and destructive fungal diseases of Brassicaceae spp. worldwide. Here, we report the draft genome sequence of strain Abra43. The assembly comprises 29 scaffolds, with an N50 value of 2.1 Mb. The assembled genome was 31,036,461 bp in length, with a G+C content of 50.85%

Role of eisosomes in the necrotrophic fungus Alternaria brassicicola

lternaria brassicicola is a fungal necrotroph responsible for the Brassicaceae dark spot disease. This fungus is a seed-borne pathogen that only affects the aerial parts of host plants causing great damages with major incidence on yield and product quality. Its transmission to seed is a major component of pathogen fitness promoting the dispersal and long-term survival of the fungus. Recently, we showed that several eisosomal protein encoding genes were overexpressed when germinated spores of A.brassicicola were exposed to osmotic and hydric stresses, which are the main constraints encountered by the fungus during the seed colonization process. MCC/eisosomes are membrane microdomains whose function is still unclear. They have been proposed to participate in the plasma membrane function by regulating the homeostasis of lipids and would promote the recruitment of specific proteins and their subsequent protection from endocytosis. Here, we deciphered the potential involvement of eisosome in pathogenicity using a reverse genetic approach by generating and characterizing mutants deficient for key eisosomal proteinencoding genes (?pil1, ?nce102, ?lsp1 and ?pil1?lsp1). In parallel, these proteins have been fused to GFP to monitor their cellular location during the plant infection and following the exposure to stresses. Depending on the mutants, the leaf and silique colonization processes were impaired by comparison to the wild-type. We also showed a strong impact of MCC/eisosome proteinmutations on the generation of appressoria-like structures