cDNA-amplified fragment length polymorphism to study the transcriptional responses of Lactobacillus rhamnosus growing in cheese-like medium.

AIMS: Lactobacillus rhamnosus is a dominant species during Parmigiano Reggiano cheese ripening and exhibits a great adaptability to unfavourable growth conditions. Gene expression of a Lact. rhamnosus, isolated from Parmigiano Reggiano cheese, grown in a rich medium (MRS) and in a cheese-like medium (CB) has been compared by a novel cDNA-amplified fragment length polymorphism (cDNA-AFLP) protocol. METHODS AND RESULTS: Two techniques, capillary and gel electrophoresis cDNA-AFLP, were applied to generate unique transcript tags from reverse-transcribed messenger RNA using the immobilization of biotinylated 3'-terminal cDNA fragments on streptavidin-coated Dynabeads. The use of three pairs of primers allowed detecting 64 genes expressed in MRS and 96 in CB. Different transcripts were observed when Lact. rhamnosus was cultured on CB and MRS. CONCLUSIONS: The cDNA-AFLP approach proved to be able to show that Lact. rhamnosus modifies the expression of a large part of genes when cultivated in CB compared with growth under optimal conditions (MRS). In particular, the profiles of the strain grown in CB were more complex probably because the cells activate different metabolic pathways to generate energy and to respond to the environmental changes. SIGNIFICANCE AND IMPACT OF STUDY: This is the first research on Lact. rhamnosus isolated from cheese and represents one of the few concerning bacterial transcriptomic analysis towards cDNA-AFLP approaches.

Comparison of phenotypic (Biolog System) and genotypic (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) methods for typing Lactobacillus plantarum isolates from raw vegetables and fruits.

The diversity of 72 isolates of Lactobacillus plantarum, previously identified from different raw vegetables and fruits, was studied based on phenotypic (Biolog System) and genotypic (randomly amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) approaches. A marked phenotypic and genotypic variability was found. Eight clusters were formed at the similarity level of 92% based on Biolog System analysis. The most numerous clusters grouped isolates apart from the original habitat. Almost all isolates fermented maltose, D,L-lactic acid, N-acetyl-D-mannosamine and dextrin, and other typical carbon sources which are prevalent in raw vegetables and fruits. None of the isolates fermented lactose and free amino acids. At high values of linkage distance, two main clusters were obtained from both UPGMA (unweighted pair group with arithmetic average) dendrograms of RAPD-PCR and AFLP analyses. The two clusters mainly separated isolates from tomatoes and carrots from those isolated from pineapples. At 2.5 linkage distance, a high polymorphism was found and several sub-clusters were formed with both analyses. In particular, AFLP allowed the differentiation of 55 of the 72 isolates of L. plantarum. The discriminatory power of each technique used was calculated through the Simpson's index of diversity (D). The values of the D index were 0.65, 0.92 and 0.99 for Biolog System, RAPD-PCR and AFLP analyses, respectively.

Whey starter for Grana Padano cheese: effect of technological parameters on viability and composition of the microbial community.

This work aimed to investigate the effects of thermal treatments and yeast extract addition on the composition of the microbial community of natural whey starters for Grana Padano cheese. Different natural whey starter samples were held at 4 degrees C for 24 h (cooling treatment), or at -20 degrees C for 24 h (freezing treatment) to evaluate the possibility of conservation, or at 54 degrees C for 1 h (heat treatment) to evaluate the effect of the temperature commonly used during curd cooking. Separately, another set of samples was enriched with 0.3, 0.5, and 1.0% (wt/vol) of yeast extract to study its effect on the growth of lactic acid bacteria (LAB) in the starter. The new approach in this study is the use of 2 culture-independent methods: length heterogeneity (LH)-reverse transcription (RT)-PCR and fluorescence microscopy. These techniques allowed us to easily, quickly, and reproducibly assess metabolically active LAB in the control and treated samples. The LH-RT-PCR technique distinguished microorganisms based on natural variations in the length of 16S rRNA amplified by RT-PCR, as analyzed by using an automatic gene sequencer. Fluorescence microscopy counts were performed by using a Live/Dead BacLight bacterial viability kit. The repeatability of LH-RT-PCR showed that this technique has great potential to reveal changes in the microbial community of natural whey starters for Grana Padano cheese. All species showed low sensitivity to cold (4 degrees C). However, after the freezing (-20 degrees C) and heating (54 degrees C) treatments, different behaviors of the species were reported, with significant changes in their viability and relative composition. Heating treatment during curd cooking profoundly affected the viability and composition of the community that remained in the cheese and that consequently modified the microbial population. At the same time, this treatment produced the selection of LAB in whey and could be considered as the first step in natural whey starter production. Addition of yeast extract stimulated the growth of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. lactis to the detriment of Lactobacillus helveticus species. Because the yeast extract altered the microflora balance, whey starter conservation at -20 degrees C and yeast extract addition cannot be suggested as technological innovations.

Fluorescence microscopy for studying the viability of micro-organisms in natural whey starters.

AIMS: The aim of this work was to study the viability and cultivability of microbial populations of different natural whey starters and to evaluate their resistance to thermal treatments (such as exposure to high or low temperatures). METHODS AND RESULTS: Twenty-three natural whey starters for Grana Padano cheese were investigated and subsequently pH measurement, plate count agar using Man-Rogasa-Sharpe (MRS) pH 5.4 agar and whey agar medium (WAM) were performed using these samples. LIVE/DEAD BacLight bacterial viability kit was used. Total count and viability of all the 23 samples were high and similar to each other (CV 20%). However, the cultivable population was lower in terms of cfu ml(-1) and number of cells per millilitre than the viable fraction and highly variable, although its count value was higher in WAM than in MRS pH 5.4. The heating (60 degrees C for 5 min and 54 degrees C for 1 h) and freezing (-20 and -80 degrees C) treatments affected the cultivability and viability of the microbial population. CONCLUSIONS: This study demonstrated the effectiveness of LIVE/DEAD BacLight bacterial viability kit, which has already been used to evaluate bacterial populations, in investigating microbial viability in a complex ecosystem such as a natural whey starter. SIGNIFICANCE AND IMPACT OF THE STUDY: The aim of this study was to quantify the presence of damaged nonviable bacterial cells in natural whey starters. The Thoma Glass is a useful method to obtain fluorescence microscopy counts to evaluate the technological performance of natural whey starters.

Plasma, urine and skin pharmacokinetics of cefepime in burns patients.

We studied the pharmacokinetics of cefepime (2 g bd) in six burns patients. Blood, urine and skin samples were collected to measure cefepime concentrations. A two-compartment model was fitted to the data. At day 1, t(1/2beta) was 2.45 +/- 0.56 h, V(ss) 0.36 +/- 0.1 L/kg, total clearance 152 +/- 25.2 mL/min, and AUC 217 +/- 34 mg*h/L. There was no statistical difference between day 1 and day 3 for any of the pharmacokinetic parameters. We demonstrated good penetration of cefepime in skin. These results show that it is not necessary to change the standard dosage of cefepime in burns patients.

[Controlled analgesia in a burn patient: fentanyl sparing effect of clonidine]. / Analgésie contrôlée par le patient brûlé: effet d'épargne de fentanyl par la clonidine.

OBJECTIVE: To determine the sparing effect of clonidine (C) on fentanyl (F) demand in burned patients under PCA. STUDY DESIGN: Prospective, randomized, double blind study versus placebo. PATIENTS: Twelve consecutive patients with mean burn surface area of 20 +/- 9%, studied between the third and the eighth day post-burn. METHODS: F was delivered by a PCA pump (bolus: 1 mg.kg-1). In the morning, burn patients received additional F (5 mg.kg-1) before hydrotherapy. After randomisation, C or placebo (P) were alternatively infused over 24 hours. Demands for F during the morning, the afternoon and the evening were noted. Pain scores were measured on a visual analogic scale. In eight patients, plasma levels of F (pF) were iteratively measured. Heart rate, arterial pressure, respiratory rate and SpO2 were monitored. RESULTS: Analgesic demands were 19.5/day under P and 9.5 under C (P < 0.0001). Pain reoccurred for pF of 4.1 under C vs 5.7 under P (P < 0.05) with same pain scores in the two groups. After a pain stimulus, pain scores were lower under F, despite lower pF (P < 0.05). Arterial pressure and heart rate were significantly lowered during the first hour of C infusion. CONCLUSION: Doses of F and pF required to reach analgesia were very high. Adding C decreases by 50% the F demand and lowers pF. Minor haemodynamic effects were observed during the first hour of C infusion.

A reference standard for plasma proteins is required for nutritional assessment of adult burn patients.

Plasma levels of visceral proteins (VP) are commonly used for evaluation of nutritional status. Low values observed in burn patients are caused by several factors including microvascular hyper-permeability and inflammatory processes. The aim of the study was to define a range of standard values specific to burn patients. Retrospective review: from days post-burn 12 to 43, four VP and three acute phase reactants (APR) were measured twice a week, in the plasma of 107 burn patients. From these data, standard' values were determined in respect with burn surface area (BSA) and post-burn time. The results were that the VP increase and APR decrease linearly during the study. Correlation between plasma proteins and BSA or post-burn day, change from protein to protein. Albumin and transferrin are less sensitive than prealbumin and especially retinol binding protein to variations of APR, but transferrin lacks of specificity. The conclusion of the study was that plasma levels of VP have to be compared to reference standard values. When levels lower than theoretical values are observed, simultaneous APR values (especially C reactive protein) have to be compared to their own reference standard, in order to separate nutritional from inflammatory effects.

[Resistance to vecuronium in burnt patients. Influence of the burnt surface on the effectiveness of the dose 95]. / Résistance au vécuronium chez le brûlé. Influence de la surface brûlée sur la dose efficace 95.

OBJECTIVE: To assess the neuromuscular blocking effect of vecuronium in adult burn patients, to draw dose-response curves, to determine the ED 95 according to burn surface area, to analyze the time course of this pattern in order to recognize the development of a resistance according to the length of postinjury period. STUDY DESIGN: Prospective open study, extending over a 12 month period. PATIENTS: Sixty-three consecutive adult burn patients in an acute phase and 13 control patients who had been thermally injured at least 500 days before their inclusion in the study. METHODS: Anaesthesia was achieved with thiopentone, fentanyl and vecuronium in patients undergoing excision and autograft surgery. Neuromuscular blockade was assessed by thumb adduction, measured by electromyography using evoked train of four responses to ulnar nerve stimulation. Dose-response curves were determined using the single dose method from only one predetermined dose of vecuronium per patient on each day of the study. Dose-response curves were compared using linear regression and ED 95 were calculated from log-probit data. RESULTS: In the control group, ED 95 was 53 mg.kg-1. Before the 7th postinjury day, patients did not differ from controls. Between the 7th and the 70th postinjury day the ED 95 increased significantly. Patients with a burn surface area (BSA) of less than 20% had a ED 95 of 69 mg.kg-1, between 20% and 40% of BSA the ED 95 was 103 mg.kg-1, between 40% and 60% BSA the ED 95 was 134 mg.kg-1 and patients with a BSA over 60% had a ED 95 at 154 mg.kg-1. The onset of action increased in all groups and was significantly different from control group. CONCLUSION: Acutely burn patients become resistant to the neuromuscular blocking effect of vecuronium. This resistance is related to the magnitude of burn injury. The mechanism of resistance is related to an increase in nicotonic acetylcholine receptors. In these patients, the dose of vecuronium must be titrated to achieve effective muscular paralysis: the correcting factor is 1.3 for a BSA under 20%, 1.9 for a BSA between 20 and 40%, 2.5 for a BSA between 40 and 60%, and 2.9 for a BSA above 60%.

[Saccharomyces boulardii fungemia in a patient with severe burns]. / Fongémie à Saccharomyces boulardii chez un brûlé grave.

A case of Saccharomyces boulardii fungaemia in a burn patient is reported. S. boulardii was administered in order to improve the digestive tolerance to enteral nutrition. After a surgical procedure, a sepsis syndrome occurred, with a white blood cell count increase, abdominal meteorism and respiratory insufficiency, associated with seven blood cultures positive to S. boulardii. Digestive translocation of the yeast seems to be the most likely origin of this fungaemia. Some fungaemias from S. boulardii have been published. However it is not usual to find such severe clinical features as in our case which leads us to reconsider the therapeutic use of S. boulardii in situations including a gastrointestinal tract suffering.

[Nutrition proteins and muscular catabolism in severely burnt patients. Comparative effects of small peptides or free amino acids]. / Protéines nutritionnelles et catabolisme musculaire chez le brûlé grave. Effets comparatifs de petits peptides ou d'acides aminés libres.

The beneficial effects of high caloric and protein enteral diet on wound healing and prevention of infection in severely burned patients is well documented. However, the relative proportion of each nutrient and especially the form of nitrogen supply have not yet been clearly established. The aim of this study was to compare, in severely burned patients, the efficiency of a partial protein hydrolysate and free amino acid formula during a 15-day enteral feeding. Twenty burned patients ranging in age from 18 to 67 years with a mean burn size of 40 +/- 12% of total body surface area, of which 31 +/- 14% was deep dermal, were studied prospectively and randomised in two groups. Group A received the free amino acid diet which was obtained by hydrolysis of the protein hydrolysate given to Group B (60% small peptides). All diets contained a nitrogen source of similar amino acid composition. Nitrogen balance was measured daily and serum protein concentrations were determined on days 0, 4, 8, 11 and 15. Anthropometric parameters, urinary 3 methylhistidine/creatinine ratio and plasma amino acid concentration were assessed on days 0, 8 and 15. Daily and cumulative nitrogen balance at D15 did not differ between the two groups. In group A, the circulating visceral proteins increased at all times of the study without decrease of acute phase reactant, whereas only transthyretin and retinol binding protein increased at D11 and D15 with a significant decrease of C-reactive protein at the same time in the other group.(ABSTRACT TRUNCATED AT 250 WORDS)

[Patient-controlled analgesia: a study of fentanyl requirements in burnt patients during the acute phase]. / Analgésie contrôlée par le patient: étude de la demande en fentanyl chez le brûlé à la phase aiguë.

This study assessed over the first post-burn week the patients' demands for opiates delivered with a PCA device, changes in opioid delivery along the day, especially during painful events and the incidence of side effects. Twenty-two patients (8 women, 14 men), mean age 33 years (+/- 12 SD), mean BSA 21% (+/- 13 SD), mean deep dermal burn 12% (+/- 11 SD) were included. Fentanyl was delivered by i.v. route using a PCA device during the first 6 days post burn. Bolus dose was fixed at 1 microgram.kg-1, lock-out time at 30 min and the 4 h limit dose at 7 micrograms.kg-1. The following data were collected every day: satisfied and non-satisfied demands, total daily dose, demands during and after dressing change period (4 h), during afternoon (10 h) and overnight periods (10 h). Heart rate (HR), respiratory rate (RR), blood pressure (BP), Paco2 and Spo2 were also monitored. The mean total daily demands were similar from D1 to D6 (28 +/- 7). Around 7 demands/day were not satisfied. The mean total daily satisfied demands were also the same: 21 +/- 5. Individual demands were over a wide range (minimum: 15, maximum: 41 demands/day for total demands and 13 and 34 respectively for the satisfied ones). Mean fentanyl delivery was 1.7 +/- 0.1 microgram.kg-1.h-1 during dressing period, 0.9 +/- 0.3 microgram.kg-1.h-1 during afternoon period (10 h) and 0.5 +/- 0.2 during overnight period (10 h). No-satisfied demands were around 4 boluses during dressing change period, 2 boluses during afternoon period, and one bolus during overnight period.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of age and gender on the in vitro serum protein binding of flecainide.

The in vitro protein binding of flecainide was studied by equilibrium dialysis in relation to serum concentrations of albumin and alpha 1-acid glycoprotein (AAG) in 22 healthy subjects of both sexes aged between 23 and 89 years. In the range of flecainide concentrations tested, protein binding of flecainide was not saturable and the percent value of the unbound fraction ranged between 0.48 and 0.68, mean value (SD) = 0.59 (+/- 0.06), without any significant difference between males and females or between young and old subjects. The flecainide unbound fraction was significantly correlated with serum albumin concentrations but not with total serum proteins or AAG concentrations.