Epiphysiolysis Type Salter I of the Medial Clavicle with Posterior Displacement: A Case Series and Review of the Literature.

Physeal fractures of the medial clavicle with posterior displacement of the metaphysis are very rare injuries, but additional injuries can be life-threatening. Due to the specific clavicular ossification process, skeletally immature patients present usually not true sternoclavicular joint (SCJ) dislocations accordingly to adults but rather displaced physeal fractures. There is no consensus in the current literature on the best treatment of this lesion. Conservative treatment is not resulting in good outcome; closed reduction is often not successful, and open reduction with internal fixation is finally required. Several methods are described for stabilizing these physeal fractures. We treated three osseous immature patients with this lesion. Due to the small dimension of the medial clavicular epiphysis, we performed in one case a transosseous figure-of-eight suture of the clavicular metaphysis towards the sternum, and in the two other cases, a transosseous suture from the clavicular metaphysis on the anterior clavicular periosteum. The latter technique avoids harm to the small epiphysis or the SCJ and minimizes the risk of retrosternal complications.

Implementation of an FTIR spectral library of 486 filamentous fungi strains for rapid identification of molds.

Filamentous fungi may cause food and feed spoilage and produce harmful metabolites to human and animal health such as mycotoxins. Identification of fungi using conventional phenotypic methods is time-consuming and molecular methods are still quite expensive and require specific laboratory skills. In the last two decades, it has been shown that Fourier transform infrared (FTIR) spectroscopy was an efficient tool for microorganism identification. The aims of this study were to use a simple protocol for the identification of filamentous fungi using FTIR spectroscopy coupled with a partial least squares discriminant analysis (PLS-DA), to implement a procedure to validate the obtained results, and to assess the transferability of the method and database. FTIR spectra of 486 strains (43 genera and 140 species) were recorded. An IR spectral database built with 288 strains was used to identify 105 different strains. It was found that 99.17% and 92.3% of spectra derived from these strains were correctly assigned at the genus and species levels, respectively. The establishment of a score and a threshold permitted to validate 80.79% of the results obtained. A standardization function (SF) was also implemented and tested on FTIR data from another instrument on a different site and permitted to increase the percentage of well predicted spectra for this set from 72.15% to 89.13%. This study confirms the good performance of high throughput FTIR spectroscopy for fungal identification using a spectral library of molds of industrial relevance.

Application of capillary electrophoresis single-stranded conformation polymorphism (CE-SSCP) analysis for identification of fungal communities in cheese.

As major contributors of the ripening process, yeasts and filamentous fungi play a fundamental role in cheese-making. Still, there is no rapid and affordable identification method available for both yeasts and filamentous fungi encountered in cheeses. In the present study, we developed a method based on CE-SSCP analysis of nuclear ribosomal DNA ITS amplicons, along with a species pattern database comprising 37 fungal species. By combining analyses of the ITS1 and ITS2 conformers, 25 out of 37 species were discriminated using CE-SSCP analysis. This reproducible and sensitive method was applied to determine the fungal community composition of 36 cheeses including blue-veined, pressed-cooked, pressed-uncooked, red-smear and surface-mould ripened cheeses. Overall, each cheese contained between 1 and 6 fungal species and 23 different species of fungi were detected including 8 yeast species, 9 filamentous species and 6 unidentified species. Comparison of the fungal diversity obtained after cloning and sequencing (rDNA ITS) versus CE-SSCP for 8 cheeses showed that CE-SSCP was at least as exhaustive as cloning and sequencing of thirty clones per cheese. In conclusion, this CE-SSCP method was an effective tool to identify the fungi present in various cheese varieties and may be of interest for the cheese industry to rapidly describe the composition of cheese fungal communities.

Differentiation and identification of filamentous fungi by high-throughput FTIR spectroscopic analysis of mycelia.

Routine identification of fungi based on phenotypic and genotypic methods can be fastidious and time-consuming. In this context, there is a constant need for new approaches allowing the rapid identification of molds. Fourier-transform infrared (FTIR) spectroscopy appears as such an indicated method. The objective of this work was to evaluate the potential of FTIR spectroscopy for an early differentiation and identification of filamentous fungi. One hundred and thirty-one strains identified using DNA sequencing, were analyzed using FTIR spectroscopy of the mycelia obtained after a reduced culture time of 48 h compared to current conventional methods. Partial least square discriminant analysis was used as a chemometric method to analyze the spectral data and for identification of the fungal strains from the phylum to the species level. Calibration models were constructed using 106 strains pertaining to 14 different genera and 32 species and were used to identify 25 fungal strains in a blind manner. Identification levels of 98.97% and 98.77% achieved were correctly assigned to the genus and species levels respectively. FTIR spectroscopy with its high discriminating power and rapidity therefore shows strong promise for routine fungal identification. Upgrading of our database is ongoing to test the technique's robustness.

Influence of Pythium oligandrum on the bacterial communities that colonize the nutrient solutions and the rhizosphere of tomato plants.

The influence exerted by the biocontrol oomycete Pythium oligandrum on the bacterial populations proliferating in the rhizosphere of tomato plants grown in a hydroponic system and in the circulating solutions is studied in the present experiment. Quantitative PCR and single-strand conformation polymorphism were used to investigate the genetic structure and dynamics of the bacterial communities colonizing the root systems and the various circulating solutions. Quantitative PCR assays showed that bacteria heavily colonized the rhizosphere of tomato plants with, however, no significant density changes throughout the cultural season (April-September). Single strand conformation polymorphism fingerprints revealed the occurrence of transient perturbations in the rhizospheric indigenous bacterial communities following P. oligandrum introduction in the root system of plants. This effect was, however, transient and did not persist until the end of the cropping season. Interestingly, the genetic structure of the bacterial microflora colonizing either the roots or the nutrient solutions evolved throughout the cropping season. This temporal evolution occurred whatever the presence and persistence of P. oligandrum in the rhizosphere. Evidence is also provided that bacterial microflora that colonize the root system are different from the ones colonizing the circulating solutions. The relationships between these 2 microflora (at the root and solution levels) are discussed.

Biodiversity of antifungal lactic acid bacteria isolated from raw milk samples from cow, ewe and goat over one-year period.

Antifungal lactic acid bacteria (ALAB) biodiversity was evaluated in raw milk from ewe, cow and goat over one year period. Lactic acid bacteria were enumerated using 8 semi-selective media, and systematically screened for their antifungal activity against 4 spoilage fungi commonly encountered in dairy products. Depending on the selective medium, between 0.05% (Elliker agar) and 5.5% (LAMVAB agar) screened colonies showed an antifungal activity. The great majority of these active colonies originated from cow (49%) and goat (43%) milks, whereas only 8% were isolated from ewe milk. Penicillium expansum was the most frequently inhibited fungus with 48.5% of colonies active against P. expansum among the 1235 isolated, followed by Mucor plumbeus with 30.6% of active colonies, Kluyveromyces lactis with only 12.1% of active colonies and Pichia anomala with 8.7% of active colonies. In the tested conditions, 94% of the sequenced active colonies belonged to Lactobacillus. Among them, targeted fungal species differed according to the Lactobacillus group, whose presence largely depended on year period and milk origin. The Lb. casei and Lb. reuteri groups, predominantly recovered in summer/fall, were overrepresented in the population targeting M. plumbeus, whereas isolates from the Lb. plantarum group, predominantly recovered in spring, were overrepresented in the population targeting K. lactis, the ones belonging to the Lb. buchneri group, predominantly recovered in spring, were overrepresented in the population targeting P. anomala. Raw milk, especially cow and goat milks from the summer/fall period appeared to be a productive reservoir for antifungal lactobacilli.

Modelling the effect of temperature, water activity and pH on the growth of Serpula lacrymans.

AIMS: To predict the risk factors for building infestation by Serpula lacrymans, which is one of the most destructive fungi causing timber decay in buildings. METHODS AND RESULTS: The growth rate was assessed on malt extract agar media at temperatures between 1.5 and 45°C, at water activity (a(w)) over the range of 0.800-0.993 and at pH ranges from 1.5 to 11.0. The radial growth rate (µ) and the lag phase (λ) were estimated from the radial growth kinetics via the plots radius vs time. These parameters were then modelled as a function of the environmental factors tested. Models derived from the cardinal model (CM) were used to fit the experimental data and allowed an estimation of the optimal and limit values for fungal growth. Optimal growth rate occurred at 20°C, at high a(w) level (0.993) and at a pH range between 4.0 and 6.0. The strain effect on the temperature parameters was further evaluated using 14 strains of S. lacrymans. The robustness of the temperature model was validated on data sets measured in two different wood-based media (Quercus robur L. and Picea abies). CONCLUSIONS: The two-step procedure of exponential model with latency followed by the CM with inflection gives reliable predictions for the growth conditions of a filamentous fungus in our study. The procedure was validated for the study of abiotic factors on the growth rate of S. lacrymans. SIGNIFICANCE AND IMPACT OF THE STUDY: This work describes the usefulness of evaluating the effect of physico-chemical factors on fungal growth in predictive building mycology. Consequently, the developed mathematical models for predicting fungal growth on a macroscopic scale can be used as a tool for risk assessment of timber decay in buildings.

Application of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces.

AIMS: To evaluate and optimize the use of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces. METHODS AND RESULTS: The resolution of DHPLC was first evaluated and optimized using a mixture of PCR amplicons of the internal transcribed spacer 2 (ITS2) region of 19 yeast reference strains representing 18 species that are common in the cheese microbiota. Sixteen of the 18 yeast species could be resolved by combining runs at temperatures of 57.5 and 59 degrees C. Then, DHPLC was used to investigate the yeast microbiota of pasteurized Maroilles, Munster and Livarot cheese surfaces by comparing their peak profiles with our reference yeast database and by collecting/sequencing of peak fractions. Debaryomyces hansenii and Geotrichum candidum for Munster and Maroilles cheeses, and Candida catenulata, Candida intermedia and G. candidum for Livarot cheese were identified using the reference database and collecting/sequencing of peak fractions. CONCLUSIONS: DHPLC technique was found to have good resolution properties and to be useful for investigating the yeast microbiota of red smear cheese surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that DHPLC is applied to study the yeast microbiota of red smear cheese surfaces.

Quantification of Penicillium camemberti and P. roqueforti mycelium by real-time PCR to assess their growth dynamics during ripening cheese.

Real-time PCR has been applied to quantify mycelium of Penicillium camemberti and Penicillium roqueforti during ripening of model cheese curd and surface mould-ripened cheeses. Total fungal DNA was first validated as an indicator of mycelial biomass in pure liquid culture and then in model curds at different stages of ripening. To imitate cheese matrix effects, DNA was extracted from curd mixed with known amounts of fresh mycelium of P. camemberti or P. roqueforti and was used as biomass standards for further quantitative real-time PCR. Mycelial mass per cheese (mg/g) was then directly obtained from fluorescence data. In model cheese curd, mycelial mass of P. camemberti increased from 2.8 at d4 to 596 mg/g at d11 whereas P. roqueforti increased from 0.3 to 6.3 mg/g during the same period. P. camemberti showed a fast development in Coulommiers from d2 to d9 (66 to 119 mg/g) and a 100-fold increase in Carré (0.85 to 85 mg/g). While mycelial biomass reached a maximum at d9 in Coulommiers, it still developed in Carré until d45. For the first time, cheese manufacturers have a powerful technique to monitor mycelial growth dynamics of their fungal cultures, which represents an important step for controlling cheese making.

Physiological traits of Penicillium glabrum strain LCP 08.5568, a filamentous fungus isolated from bottled aromatized mineral water.

Penicillium glabrum is a ubiquitous fungus distributed world wide. This fungus is a frequent contaminant in the food manufacturing industry. Environmental factors such as temperature, water activity and pH have a great influence on fungal development. In this study, a strain of P. glabrum referenced to as LCP 08.5568, has been isolated from a bottle of aromatized mineral water. The effects of temperature, a(w) and pH on radial growth rate were assessed on Czapeck Yeast Agar (CYA) medium. Models derived from the cardinal model with inflection [Rosso et al., 1993 An unexpected correlation between cardinal temperatures of microbial growth highlighted by a new model. J. Theor. Bio. 162, 447-463.] were used to fit the experimental data and determine for each factor, the cardinal parameters (minimum, optimum and maximum). Precise characterisation of the growth conditions for such a fungal contaminant, has an evident interest to understand and to prevent spoilage of food products.

Isolation and analysis of differentially expressed genes in Penicillium glabrum subjected to thermal stress.

Penicillium glabrum is a filamentous fungus frequently involved in food contamination. Numerous environmental factors (temperature, humidity, atmospheric composition, etc.) or food characteristics (water activity, pH, preservatives, etc.) could represent potential sources of stress for micro-organisms. These factors can directly affect the physiology of these spoilage micro-organisms: growth, conidiation, synthesis of secondary metabolites, etc. This study investigated the transcriptional response to temperature in P. glabrum, since this factor is one of the most important for fungal growth. Gene expression was first analysed by using suppression subtractive hybridization to generate two libraries containing 445 different up- and downregulated expressed sequence tags (ESTs). Expression of these ESTs was then assessed for different thermal stress conditions, with cDNA microarrays, resulting in the identification of 35 and 49 significantly up- and downregulated ESTs, respectively. These ESTs encode heat-shock proteins, ribosomal proteins, superoxide dismutase, trehalose-6-phosphate synthase and a large variety of identified or unknown proteins. Some of these may be molecular markers for thermal stress response in P. glabrum. To our knowledge, this work represents the first study of the transcriptional response of a food spoilage filamentous fungus under thermal stress conditions.

Characterization of Bacillus and Pseudomonas strains with suppressive traits isolated from tomato hydroponic-slow filtration unit.

Bacillus and Pseudomonas spp. are known to be involved in plant pathogenic fungi elimination during the slow filtration process used in tomato soilless cultures. We isolated 6-8 strains of both Bacillus and Pseudomonas from the top, middle, and bottom sections of filters and identified them after 16S rDNA sequencing. Four Pseudomonas strains were identified as Pseudomonas fulva, 5 as Pseudomonas plecoglossicida, and 12 as Pseudomonas putida. The use of specific oligonucleotide polymerase chain reaction primer sets designed from gyrB gene sequences additionally permitted the identification of 17 Bacillus cereus and 3 Bacillus thuringiensis strains. Ribotyping with EcoRI pointed out an important polymorphism within Bacillus and Pseudomonas strains. Molecular characterization did not reveal a correlation between the location of isolates within the filter (top, middle, or bottom) and bacterial identification or riboclusters. Functional aspects assessed by community-level physiological profiling showed marked phenotypic differences between Pseudomonas communities isolated from the top and bottom filter layers; differences were lower between Bacillus communities of different layers and far less noticeable between mixed communities of Bacillus and Pseudomonas. These strains were tested for several suppressive activities. Conversely to most Bacillus, the majority of Pseudomonas strains were auxin producers and promoted the growth of tomato plantlet roots. On the other hand, only Bacillus strains displayed antagonistic activities by inhibiting the growth of pathogenic fungi frequently detected in soilless cultures. Siderophores were produced by nearly all bacteria, but at higher amounts by Pseudomonas than Bacillus strains. The biocontrol agent potentiality of certain strains to optimize the slow filtration process and to promote the suppressive potential of nutrient solution is discussed.

Caloranaerobacter azorensis gen. nov., sp. nov., an anaerobic thermophilic bacterium isolated from a deep-sea hydrothermal vent.

A thermophilic, anaerobic, chemo-organotrophic bacterium, designated MV1087T, was isolated from a deep-sea hydrothermal chimney sample collected from the Mid-Atlantic Ridge. The cells were straight, motile and stained gram-negative. Growth was observed from 45 to 65 degrees C, with an optimum around 65 degrees C. No growth was observed at 40 or 70 degrees C. Growth was observed from pH 5.5 to 9.0 and the optimum pH was around 7. The salinity range for growth was 10-100 g sea salt l(-1) (corresponding to 6.5-65 g NaCl l(-1)) with an optimum at 30 g sea salt l(-1) (20 g NaCl l(-1)). Strain MV1087T was heterotrophic, able to ferment proteinaceous substrates, such as brain/heart infusion and gluten, and carbohydrates, such as glucose, xylan and starch. The DNA G+C content was 27 mol%. Phylogenetic analyses using 16S rDNA sequences indicated that strain MV1087T belonged to cluster XII of the Clostridium subphylum. Due to its phenotypic and genotypic characteristics, isolate MV1087T is proposed as a novel species of a new genus, Caloranaerobacter azorensis gen. nov., sp. nov. The type strain is MV1087T (= CNCM I-2543T = DSM 13643T).

Marinitoga camini gen. nov., sp. nov., a rod-shaped bacterium belonging to the order Thermotogales, isolated from a deep-sea hydrothermal vent.

A thermophilic, anaerobic, chemo-organotrophic sulfur-reducing bacterium, designated MV1075T, was isolated from a deep-sea hydrothermal chimney sample collected on the Mid-Atlantic Ridge. Cells were rod-shaped with a sheath-like outer structure, motile with polar flagella and stained Gram-negative. They appeared singly, in pairs or in short chains. The temperature range for growth was 25-65 degrees C, with an optimum at 55 degrees C. Growth was observed from pH 5 to pH 9, and the optimum pH was around 7. The salinity range for growth was 15-70 g sea salt l(-1) (corresponding to 10-45 g NaCl l(-1)), with an optimum at 30 g l(-1) (20 g NaCl l(-1)). The isolate was able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates. Sulfur was not necessary for growth. Growth was inhibited by H2, but, in presence of sulfur, this inhibition was removed and H2S was produced. The G+C content of the genomic DNA was 29 mol %. Phylogenetic analyses of the 16S rRNA gene located the strain within the order Thermotogales, in the domain Bacteria. On the basis of 16S rDNA sequence comparisons, in combination with morphological and physiological characteristics, it is proposed that the isolate should be described as a novel species of a new genus, Marinitoga gen. nov., of which Marinitoga camini sp. nov. is the type species. The type strain is MV1075T (= CNCM 1-2413T = DSM 13578T).

Microbial diversity in smoked salmon examined by a culture-independent molecular approach--a preliminary study.

Microbial biodiversity in sliced vacuum-packed cold smoked salmon was investigated using culture-independent molecular biology techniques. Sliced smoked salmon was stored for 25 days after being packed at 4 degrees C. DNA was extracted from sliced vacuum-packed cold smoked salmon. PCR DNA amplification were carried out using universal eubacterial primers corresponding to Escherichia coli 16S rRNA gene. 16S rRNA genes were amplified, cloned in E. coli and compared using Amplification Ribosomal DNA Restriction Analysis (ARDRA). 106 clones were studied and classified into 13 Operational Taxonomic Units (OTUs). Sequences obtained to describe those 13 OTUs were compared to GenBank data. They indicated the presence of Vibrio species. Enterobacteraceae and also marine psychrophilic clones related to Alteromonas macleodii, which were not encountered within cultures, but no Gram-positive species have been obtained. Those results indicate that bias in description of microbial diversity may be encountred in both molecular and cultural techniques.

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.

Pyrococcus glycovorans sp. nov., a hyperthermophilic archaeon isolated from the East Pacific Rise.

A hyperthermophilic archaeon, strain AL585T, was isolated from a deep-sea hydrothermal vent located on the East Pacific Rise at latitude 13 degrees N and a depth of 2650 m. The isolate was a strictly anaerobic coccus with a mean cell diameter of 1 micron. The optimum temperature, pH and concentration of sea salt for growth were 95 degrees C, 7.5 and 30 g l-1. Under these conditions, the doubling time and cell yield were 0.5 h and 5 x 10(8) cells ml-1. Strain AL585T grew preferentially in media containing complex proteinaceous carbon sources, glucose and elemental sulfur. The G + C content of the DNA was 47 mol%. Sequencing of the 16S rDNA gene showed that strain AL585T belonged to the genus Pyrococcus and was probably a new species. This was confirmed by total DNA hybridization. Consequently, this strain is described as a new species, Pyrococcus glycovorans sp. nov.

Unpredictable response to vasodilator therapy in primary pulmonary hypertension.

A 49-year-old man with severe primary pulmonary hypertension unresponsive to multiple vasodilators showed beneficial response to intravenous (i.v.) isoproterenol with significant decrease in the pulmonary vascular resistance and increase in the cardiac output. However, the patient became dependent on i.v. isoproterenol and eventually died suddenly following an episode of bradyarrhythmia. Autopsy confirmed severe primary pulmonary hypertension. As the response to vasodilator therapy can be unpredictable in PPH, sequential hemodynamic evaluation of drugs is necessary for finding an optimal therapeutic agent.

Purification, molecular properties and specificity of a thermoactive and thermostable proteinase from Pyrococcus abyssi, strain st 549, hyperthermophilic archaea from deep-sea hydrothermal ecosystem.

A protease was isolated and purified from the supernatant of a culture of hyperthermophilic archaebacteria: Pyrococcus abyssi strain st 549. Purification consisted of three chromatographic steps. The enzyme purification yield was 4% and the purification factor 890. This protease is a seryl-protease hydrolyzing proteins and peptides with a preference for cleavage at the aromatic and hydrophobic residues in P1 and P'1 positions. Its activity is optimal at 95 degrees C and at pH 9. The electrophoretic mobility of the protease observed by zymogram suggests that it can adopt several oligomer forms. Three of them predominate displaying apparent molecular masses of 150, 105 and 60 kDa. Interdependence of the observed bands was revealed by changing the denaturation conditions of the samples (temperature, SDS concentration) before electrophoresis.

Vibrio diabolicus sp. nov., a new polysaccharide-secreting organism isolated from a deep-sea hydrothermal vent polychaete annelid, Alvinella pompejana.

A deep-sea, facultatively anaerobic, heterotrophic, mesophilic new organism was isolated from the polychaete annelid Alvinella pompejana collected from a deep-sea hydrothermal field in the East Pacific Rise. On the basis of phenotypic characteristics, phylogenetic analyses, and DNA-DNA relatedness, this organism was identified as a new species of the genus Vibrio, for which the name Vibrio diabolicus is proposed. In batch cultures in the presence of glucose, this organism produced an innovative exopolysaccharide. This polymer had high contents of both uronic acids and hexosamines and was similar to other polysaccharides with interesting biological activities.