Diversity and efficiency of bradyrhizobium strains isolated from soil samples collected from around sesbania virgata roots using cowpea as trap species

The genetic diversity of ten Bradyrhizobium strains was evaluated for tolerance to high temperatures, to different salinity levels and for the efficiency of symbiosis with cowpea plants (Vigna unguiculata (L.) Walp.). Eight of these strains were isolated from nodules that appeared on cowpea after inoculation with suspensions of soil sampled from around the root system of Sesbania virgata (wand riverhemp) in ecosystems of South Minas Gerais. The other two strains used in our analyses as references, were from the Amazon and are currently recommended as cowpea inoculants. Genetic diversity was analyzed by amplifying repetitive DNA elements with the BOX primer, revealing high genetic diversity with each strain presenting a unique band profile. Leonard jar assays showed that the strains UFLA 03-30 and UFLA 03-38 had the highest N2-fixing potentials in symbiosis with cowpea. These strains had more shoot and nodule dry matter, more shoot N accumulation, and a higher relative efficiency than the strains recommended as inoculants. All strains grew in media of pH levels ranging from 4.0 to 9.0. The strains with the highest N2-fixing efficiencies in symbiosis with cowpea were also tolerant to the greatest number of antibiotics. However, these strains also had the lowest tolerance to high salt concentrations. All strains, with the exceptions of UFLA 03-84 and UFLA 03-37, tolerated temperatures of up to 40 ºC. The genetic and phenotypic characteristics of the eight strains isolated from soils of the same region were highly variable, as well as their symbiotic efficiencies, despite their common origin. This variability highlights the importance of including these tests in the selection of cowpea inoculant strains.

Allergies alimentaires rares [Rare food allergies]

A limited number of foods explain the majority of food allergies. These allergies can be due to a weak allergenicity (garlic, onion, potato), or a weak (or increasing) exposure to emergent food allergens which can be imported (exotic fruits), or recently introduced (lupin, buckwheat, sesame, inulin) or modified by the industry (lysats, lecithins, traces of antibiotics, caseinates, molds, dust mite). Others are in relation with rarer cross-reactivity food allergy syndrome (Apiaceae-Compositae-mugwort syndrome, egg-bird syndrome, cat epithelium-pork meat syndrome). Others are rarely identified, because the food is masked (pepper, basilic). We illustrate rare cases of food allergy and discuss the diagnostic management which is based on a meticulous patient history. Un nombre restreint d'aliments explique la majorité des allergies alimentaires. Les allergies alimentaires rares sont dues à une faible allergénicité (ail, oignon, pomme de terre) ou à une exposition faible ou croissante à des aliments émergents, importés (fruits exotiques), introduits (lupin, sarrasin, sésame, inuline), ou modifiés par l'industrie (lysats, lécithines, traces d'antibiotiques, caséinates, moisissures, acariens). D'autres sont en relation avec des croisements d'allergènes rares (syndrome croisé ombellifères-composées-armoise, syndrome oeuf-oiseau, syndrome épithélium de chat-viande de porc). D'autres enfin sont rarement identifiées, car l'allergène est masqué (poivre, basilic). Nous décrivons des cas rares illustratifs et rappelons la démarche diagnostique qui s'appuie sur une anamnèse minutieuse.

Influence of plant species on disease suppression by Pseudomonas fluorescens strain CHA0 with enhanced antibiotic production

Introduction of the recombinant cosmid pME3090 into Pseudomonas fluorescens strain CHAO, a good biocontrol agent of various diseases caused by soilborne pathogens, increased three- to five-fold the production of the antibiotic metabolites pyoluteorin (Pit) and 2,4-diacetylphlorogIucinol (Phi) in vitro. Strain CHAO/pME3090 also overproduced Pit and Phi in the rhizosphere of wheat infected or not infected with Pythium ultimum. The biocontrol activity of the wild-type and recombinant Straitis was compared using various plant pathogen-host combinations in a gnotobiotic system. Antibiotic overproduction affected neither the protection of wheat against P. ultimum and Gaeumannomyces graminis var. tritici nor the growth of wheat plants. In contrast, strain CHA0/pME3090 showed an increased capacity to protect cucumber against Fusarium oxysporum f. sp. cucumerinum and Phomopsis sclerotioides, compared with the wild-type strain CHAO, The antibiotic overproducing strain protected tobacco roots significantly better against Thielaviopsis basicola than the wild-type strain but drastically reduced the growth of tobacco plants and was also toxic to the growth of sweet com. On King's B agar and on malt agar, the recombinant strain CHA0/pME3090 inhibited all pathogens more than did the parental strain CHAO. Synthetic Pit and Phi were toxic to all fungi tested. Tobacco and sweet com were more sensitive to synthetic Pit and Phi than were cucumber and wheat. There was no correlation between the sensitivity of the pathogens to the synthetic antibiotics and the degree of disease suppression by strain CHAO pME3090. However, there was a correlation between the sensitivity of the plants and the toxicity of the recombinant strain. We conclude that the plant species rather than the pathogen determines whether cosmid pME3090 in P. fluorescens strain CHAO leads to improved disease suppression.

Prise en charge initiate du sepsis sévère et du choc septique [Management of severe sepsis and septic shock].

Severe sepsis and septic shock are systemic manifestations of the host response to infection. Mortality remains high despite advances in pathophysiological knowledge. Hemodynamic and respiratory management is largely supportive, while early antibiotics administration and source of infection's control are crucial for patient outcome. We review the principles guiding the initial management of these patients in emergency situation.

Impact of Pseudomonas fluorescens strain CHA0 and a derivative with improved biocontrol activity on the culturable resident bacterial community on cucumber roots

Information on the effects of released wild-type or genetically engineered bacteria on resident bacterial communities is important to assess the potential risks associated with the introduction of these organisms into agroecosystems. The rifampicin-resistant biocontrol strain Pseudomonas fluorescens CHA0-Rif and its derivative CHA0-Rif/pME3424, which has improved biocontrol activity and enhanced production of the antibiotics 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt), were introduced into soil microcosms and the culturable bacterial community developing on cucumber roots was investigated 10 and 52 days later. The introduction of either of the two strains led to a transiently enhanced metabolic activity of the bacterial community on glucose dimers and polymers as measured with BIOLOG GN plates, but natural succession between the two sampling dates changed the metabolic activity of the bacterial community more than did the inoculants. The introduced strains did not significantly affect the abundance of dominant genotypic groups of culturable bacteria discriminated by restriction analysis of amplified 16S rDNA of 2500 individual isolates. About 30-50% of the resident bacteria were very sensitive to Phl and Plt, but neither the wild-type nor CHA0-Rif/pME3424 changed the proportion of sensitive and resistant bacteria in situ. In microcosms with a synthetic bacterial community, both biocontrol strains reduced the population of a strain of Pseudomonas but did not affect the abundance of four other bacterial strains including two highly antibiotic-sensitive isolates. We conclude that detectable perturbations in the metabolic activity of the resident bacterial community caused by the biocontrol strain CHA0-Rif are (i) transient, (ii) similar for the genetically improved derivative CHA0-Rif/pME3424 and (iii) less pronounced than changes in the community structure during plant growth.

Péritonite infectieuse en dialyse péritonéale: une complication trop redoutée [Infectious peritonitis in peritoneal dialysis: an over-emphasized complication].

Peritoneal dialysis is an extrarenal epuration modality which uses physiological properties of peritoneum as a dialysis membrane. Despite the improvement of peritoneal dialysis techniques in the last ten years, peritonitis remains one of the most redoubt complications. Peritonitis may sometimes lead to technical failures, which need catheter removing, but rarely lead to death. Our retrospective study at the dialysis center of CHUV has analyzed factors which can predict this kind of complication. It calculates peritonitis rate and median peritonitis free-survival for different groups of patients. It also describes causatives organisms and their sensitivity to antibiotics.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Effect of elective antibiotic therapy on resting energy expenditure and inflammation in cystic fibrosis.

Cystic fibrosis (CF) patients often present with malnutrition which may partly be due to increased resting energy expenditure (REE) secondary to inflammation. Both REE and tumour necrosis factor-alpha (TNF-alpha), as other markers of inflammation, are elevated during respiratory exacerbations and decrease after antibiotic treatment. However, the effect of antibiotic therapy on REE and inflammation in patients without respiratory exacerbation is not known. The aim of our study was to determine the effect of such an elective antibiotic therapy on REE, TNF-alpha, and other serum markers of inflammation. Twelve CF patients 5F/7M, age 15.9 +/- 6.1 years, weight for height ratio 89 +/- 8% without clinically obvious exacerbation and treated by intravenous antibiotics were studied. Both before (D0) and after (D14) treatment, pulmonary function tests were performed. REE was measured by indirect calorimetry and blood taken to measure inflammation parameters. Body weight increased by 1.1 kg from D0 to D14 (P < 0.001), composed of 0.3 kg fat mass and 0.8 kg fat-free mass (FFM). The forced expiratory volume at 1 s increased from 43 +/- 15% of predicted at D0 to 51 +/- 15% of predicted at D14 (P < 0.01). Mean REE was 41.1 +/- 7.6 kcal/kg FFM per day at D0 and did not change significantly at D14 (40.6 +/- 8.5 kcal/kg FFM per day). Serum markers of inflammation decreased from D0 to D14: C-reactive protein 17 +/- 17 mg/l to 4 +/- 7 mg/l (P < 0.05), elastase 62 +/- 29 microg/l to 45 +/- 18 microg/l (P < 0.02), orosomucoid acid 1.25 +/- 0.11 g/l to 0.80 +/- 0.15 g/l (P < 0.001), and TNF-alpha 37 +/- 14 pg/ml to 29 +/- 6 pg/ml (P = 0.05). Individual values showed a correlation between changes in REE and in TNF-alpha (P < 0.02). The contribution of inflammation to energy expenditure is possible but appears to be minimal in cystic fibrosis patients treated by antibiotics on a regular basis in the absence of clinically obvious exacerbation.

Therapy of mild to moderate luminal Crohn's disease.

The management of luminal Crohn's disease, the most common form of initial presentation of the disease, depends on the location and the severity of the lesions. Mild to moderate disease represents a relatively large proportion of patients with a first flare of luminal disease, which may also be associated with perianal disease. As quality of life of these patients correlates with disease activity, adequate therapy is a central goal of the overall patient management. Treatment options include mainly sulfasalazine, budesonide and systemic steroids, while the role of mesalazine and antibiotics remains controversial. The role of biological therapies in mild to moderate disease has not been thoroughly evaluated and will not be discussed here.

Regulation and glucocorticoid-independent induction of lipocortin I in cultured astrocytes.

Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP-, calcium ionophore A23187-, or tetradecanoyl phorbol acetate (TPA)-stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC-mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2 (PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2-3-week-old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum- and GC-free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative antibiogram as a typing method for the prospective epidemiological surveillance and control of MRSA: comparison with molecular typing.

OBJECTIVE: Evaluation of the quantitative antibiogram as an epidemiological tool for the prospective typing of methicillin-resistant Staphylococcus aureus (MRSA), and comparison with ribotyping. METHODS: The method is based on the multivariate analysis of inhibition zone diameters of antibiotics in disk diffusion tests. Five antibiotics were used (erythromycin, clindamycin, cotrimoxazole, gentamicin, and ciprofloxacin). Ribotyping was performed using seven restriction enzymes (EcoRV, HindIII, KpnI, PstI, EcoRI, SfuI, and BamHI). SETTING: 1,000-bed tertiary university medical center. RESULTS: During a 1-year period, 31 patients were found to be infected or colonized with MRSA. Cluster analysis of antibiogram data showed nine distinct antibiotypes. Four antibiotypes were isolated from multiple patients (2, 4, 7, and 13, respectively). Five additional antibiotypes were isolated from the remaining five patients. When analyzed with respect to the epidemiological data, the method was found to be equivalent to ribotyping. Among 206 staff members who were screened, six were carriers of MRSA. Both typing methods identified concordant of MRSA types in staff members and in the patients under their care. CONCLUSIONS: The quantitative antibiogram was found to be equivalent to ribotyping as an epidemiological tool for typing of MRSA in our setting. Thus, this simple, rapid, and readily available method appears to be suitable for the prospective surveillance and control of MRSA for hospitals that do not have molecular typing facilities and in which MRSA isolates are not uniformly resistant or susceptible to the antibiotics tested.

THE RESURRECTION PLANT TRIPOGON SPICATUS (POACEAE) HARBORS A DIVERSITY OF PLANT GROWTH PROMOTING BACTERIA IN NORTHEASTERN BRAZILIAN CAATINGA

Plant species that naturally occur in the Brazilian Caatinga(xeric shrubland) adapt in several ways to these harsh conditions, and that can be exploited to increase crop production. Among the strategic adaptations to confront low water availability, desiccation tolerance stands out. Up to now, the association of those species with beneficial soil microorganisms is not well understood. The aim of this study was to characterize Tripogon spicatusdiazotrophic bacterial isolates from the Caatingabiome and evaluate their ability to promote plant growth in rice. Sixteen bacterial isolates were studied in regard to their taxonomic position by partial sequencing of the 16S rRNA gene, putative diazotrophic capacity, in vitro indole-acetic acid (IAA) production and calcium phosphate solubilization, metabolism of nine different C sources in semi-solid media, tolerance to different concentrations of NaCl to pHs and intrinsic resistance to nine antibiotics. Finally, the ability of the bacterial isolates to promote plant growth was evaluated using rice (Oryza sativa) as a model plant. Among the 16 isolates evaluated, eight of them were classified as Enterobacteriaceae members, related to Enterobacter andPantoeagenera. Six other bacteria were related toBacillus, and the remaining two were related toRhizobiumand Stenotrophomonas.The evaluation of total N incorporation into the semi-solid medium indicated that all the bacteria studied have putative diazotrophic capacity. Two bacteria were able to produce more IAA than that observed for the strain BR 11175Tof Herbaspirillum seropedicae.Bacterial isolates were also able to form a microaerophilic pellicle in a semi-solid medium supplemented with different NaCl concentrations up to 1.27 mol L-1. Intrinsic resistance to antibiotics and the metabolism of different C sources indicated a great variation in physiological profile. Seven isolates were able to promote rice growth, and two bacteria were more efficient than the reference strainAzospirillum brasilense, Ab-V5. The results indicate the potential of T. spicatus as native plant source of plant growth promoting bacteria.

Multiplex PCR of sonication fluid accurately differentiates between prosthetic joint infection and aseptic failure.

OBJECTIVE: Cultures have limited sensitivity in the diagnosis of prosthetic joint infection (PJI), especially in low-grade infections. We assessed the value of multiplex PCR in differentiating PJI from aseptic failure (AF). METHODS: Included were patients in whom the joint prosthesis was removed and submitted for sonication. The resulting sonication fluid was cultured and investigated by multiplex PCR, and compared with periprosthetic tissue culture. RESULTS: Among 86 explanted prostheses (56 knee, 25 hip, 3 elbow and 2 shoulder prostheses), AF was diagnosed in 62 cases (72%) and PJI in 24 cases (28%). PJI was more common detected by multiplex PCR (n=23, 96%) than by periprosthetic tissue (n=17, 71%, p=0.031) or sonication fluid culture (n=16, 67%, p=0.016). Among 12 patients with PJI who previously received antibiotics, periprosthetic tissue cultures were positive in 8 cases (67%), sonication fluid cultures in 6 cases (50%) and multiplex PCR in 11 cases (92%). In AF cases, periprosthetic tissue grew organisms in 11% and sonication fluid in 10%, whereas multiplex PCR detected no organisms. CONCLUSIONS: Multiplex PCR of sonication fluid demonstrated high sensitivity (96%) and specificity (100%) for diagnosing PJI, providing good discriminative power towards AF, especially in patients previously receiving antibiotics.

Empirical antifungal therapy in neutropaenic cancer patients with persistent fever

Invasive fungal infections are frequent and severe complications in leukaemic patients with prolonged neutropaenia. Empirical antifungal therapy has become the standard of care in patients with persistent fever despite treatment with broad-spectrum antibiotics. For decades amphotericin B deoxycholate has been the sole option for empirical antifungal therapy. Recently, several new antifungal agents became available. The choice of the most appropriate drug should be guided by efficacy and safety criteria. The recommendations from the First European Conference on Infections in Leukaemia (ECIL-1) on empirical antifungal therapy in neutropaenic cancer patients with persistent fever have been developed by an expert panel after assessment of clinical practices in Europe and evidence-based review of the literature. Many antifungal regimens can now be recommended for empirical therapy in neutropaenic cancer patients. However, persistent fever lacks specificity for initiation of therapy. Development of empirical and pre-emptive strategies using new clinical parameters, laboratory markers and imaging techniques for early diagnosis of invasive mycoses are needed.

The Effect of Inoculum Size on Selection of In Vitro Resistance to Vancomycin, Daptomycin, and Linezolid in Methicillin-Resistant Staphylococcus aureus.

OBJECTIVES: The inoculum effect (IE) is an increase in the minimum inhibitory concentration (MIC) at high bacterial densities. The effect of three inoculum sizes on the selection of resistance to vancomycin, daptomycin, and linezolid was investigated in methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Low (10(4) CFU/ml), medium (10(6) CFU/ml), and high (10(8) CFU/ml) inocula of MRSA were exposed to twofold increasing concentrations of either drug during 15 days of cycling. MICs for low (MICL), medium (MICM), and high (MICH) inocula were determined daily. Conventional MICs were measured at days 1, 5, 10, and 15. Experiments were performed in triplicate. RESULTS: At the beginning of the experiment a small IE was observed for vancomycin (MICL=1 μg/ml, MICM=1-2 μg/ml, and MICH=2 μg/ml) and a significant IE for daptomycin (MICL=0.25 μg/ml, MICM=0.25-0.5 μg/ml, and MICH=2 μg/ml). Linezolid exhibited no IE at low and medium inocula (MICL=1 μg/ml and MICM=1-2 μg/ml), but with the high inoculum, concentrations up to 2,048 μg/ml did not fully inhibit visual growth. During cycling, increase of MIC was observed for all antibiotics. At day 15, MICL, MICM, and MICH of vancomycin were 2-4, 4-8, and 4-16 μg/ml and of daptomycin were 0.5-2, 8-128, and 64-256 μg/ml, respectively. MICL and MICM of linezolid were 1 and 2-4 μg/ml, respectively. Conventional MICs showed vancomycin and daptomycin selection of resistance since day 5 depending on the inocula. No selection of linezolid resistance was observed. CONCLUSIONS: Our results showed the importance of the inoculum size in the development of resistance. Measures aimed at lowering the inoculum at the site of infection should be used whenever possible in parallel to antimicrobial therapy.