The goby fish Sicydium spp. as valuable sentinel species towards the chemical stress in freshwater bodies of West Indies.

Implementation of the European Water Framework Directive in tropical areas such as the French West Indies (FWI) requires to select relevant aquatic sentinel species for investigating the ecological status of surface waters. The present work aimed to study the biological response of the widespread fish Sicydium spp. towards river chemical quality in Guadeloupe island through a set of proper biomarkers. During a 2-year survey, the hepatic EROD activity, the micronucleus formation and the level of primary DNA strand breaks in erythrocytes were measured respectively as an enzymatic biomarker of exposure and genotoxicity endpoints in fish living upstream and downstream of two chemically-contrasted rivers. Hepatic EROD activity was shown to be variable along the time but always significantly higher in fish from the most contaminated river (Rivière aux Herbes) compared to the low contaminated one (Grande Rivière de Vieux-Habitants). Fish size did not influence EROD activity. Female fish exhibited a lower EROD activity compared to males depending on the catching period. We observed significant temporal variation in micronucleus frequency and primary DNA damage level measured in fish erythrocytes that did not depend on the fish size. Micronucleus frequency and to a lesser extent DNA damage were significantly higher in fish from the Rivière aux Herbes compared to the Grande Rivière de Vieux-Habitants. Our results argue for the interest of using Sicydium spp. as sentinel species to assess river quality and chemical pressures in FWI.

Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A.

This is a pioneering study in the ecotoxicological assessment of immunotoxic effects of the three selected drugs of concern to a freshwater gastropod species. Lymnaea stagnalis was exposed in the laboratory for 3 days to three drugs used for immune systems: diclofenac (nonsteroidal anti-inflammatory drug), cyclophosphamide (anti-cancer immunosuppressive drug) or cyclosporine A (anti-xenograft immunosuppressive drug). Exposure ranges included environmental realistic (1-10µgL-1) and therapeutic concentrations (100-1000µgL-1). At the end of exposure times, the immune parameters of individual snails were measured: hemocyte density and viability, hemocyte phagocytosis capacity and hemocyte-related oxidative activities (basal and NADPH-oxidase stimulated with zymosan particles). Diclofenac and cyclosporine A induced immune responses, although the effects were not strong. No immunosuppression was observed. Such subtle immunomodulations bring further interrogations regarding their long-term immunotoxicity and possible resulting tradeoffs with life-history traits. On the other hand, the prodrug cyclophosphamide did not induce significant immune responses. Since metabolism pathways differ greatly between vertebrates and invertebrates, this study also suggests that relevant vertebrate metabolites should be included in the immunotoxicity assessment of pharmaceuticals in non-target invertebrate species. Finally, the possible interactive effects of these pharmaceuticals sharing similar modes of action or effects features should also be explored.

Organic solvents impair life-traits and biomarkers in the New Zealand mudsnail Potamopyrgus antipodarum (Gray) at concentrations below OECD recommendations.

Potamopyrgus antipodarum is a gastropod mollusk proposed for use in the development of reproduction tests within the Organization for Economic Cooperation and Development (OECD). Numerous chemicals, including endocrine disrupters, are relatively water-insoluble, and water-miscible solvents are currently used for testing them. OECD recommends a maximum concentration of 100 µll(-1). As several studies highlighted effects of lower concentrations of solvents, this study assessed the effects of 20 µll(-1) acetone, ethanol, methanol and dimethylsulfoxide (DMSO) on juvenile and adult snails during 42 days. Ethanol decreased juvenile growth, while acetone increased the rate of embryonic development. All solvents increased estradiol-like levels in adult snails. DMSO only increased mRNA expression of vitellogenin-like gene, while acetone, ethanol and methanol decreased mRNA expression of three nuclear receptor (estrogen receptor-like, ecdysone-induced protein and chicken ovalbumin upstream promoter transcription factor) genes as well as of genes encoding proteins involved in genomic (prohibitin-2) and non-genomic (striatin) pathways of estrogens activity in vertebrates. This study highlights the confounding effects of low concentrations of solvents and recommends avoiding their use. Where solvent use is inevitable, their concentrations and type should be investigated for suitability for the measured endpoints prior to use in chemical testing strategies.

One-year monitoring of core biomarker and digestive enzyme responses in transplanted zebra mussels (Dreissena polymorpha).

A 12-month active biomonitoring study was performed in 2008-2009 on the Vesle river basin (Champagne-Ardenne, France) using the freshwater mussel Dreissena polymorpha as a sentinel species; allochthonous mussels originating from a reference site (Commercy) were exposed at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle river basin. Selected core biomarkers (acetylcholinesterase (AChE) activity, glutathione-S transferase (GST) activity, metallothionein concentration), along with digestive enzyme activities (amylase, endocellulase) and energy reserve concentrations (glycogen, lipids), were monitored throughout the study in exposed mussels. At the Fismes and Ardre sites (downstream basin), metallic and organic contamination levels were low but still high enough to elicit AChE and GST activity induction in exposed mussels (chemical stress); besides, chemical pollutants had no apparent deleterious effects on mussel condition. At the Bouy and Sept-Saulx sites (upstream basin), mussels obviously suffered from adverse food conditions which seriously impaired individual physiological state and survival (nutritional stress); food scarcity had however no apparent effects on core biomarker responses. Digestive enzyme activities responded to both chemical and nutritional stresses, the increase in energy outputs (general adaptation syndrome-downstream sites) or the decrease in energy inputs (food scarcity-upstream sites) leading to mid- or long-term induction of digestive carbohydrase activities in exposed mussels (energy optimizing strategy). Complex regulation patterns of these activities require nevertheless the use of a multi-marker approach to allow data interpretation. Besides, their sensitivity to natural confounding environmental factors remains to be precised.

Non destructive in vivo measurement of ethoxyresorufin biotransformation by zebrafish prolarva: development and application.

Biotransformation enzymatic activities, such as the cytochrome P450 one, have been used as biomarkers for pollution assessment for a long time. Nevertheless, such biochemical tools are destructive processes, because they are performed on fish liver or total larvae homogenates. Moreover, the adaptation of this bioassay to some fish larvae, like Danio rerio ones, is ineffective because it needs a lot of organisms. We thus developed an original, nondestructive method to detect the induction of a biotransformation activity in the prolarva of the fish, Danio rerio, exposed to chemicals. This methodology is based on the assessment of EROD activity, by measurement in the culture medium of the fluorescence increase due to the excretion of resorufin by fish during an ethoxyresorufin exposure. After exposure of fish embryos to known inducers (BaP and beta-naphtoflavone), the prolarvae were exposed to the substrate (ethoxyresorufin), and the kinetic of the fluorescence increase was measured. A dose-effect relationship was observed, with a three to five fold increase of EROD basal activity. This methodology also allowed us to compare between EROD activity induction by different environmental samples. The proposed methodology thus allows to perform a simple, sensitive, and reproducible microbiotest for the detection of sublethal concentrations of AhR chemical inducers in environmental samples.

Antibiotic resistance rates and phenotypes among isolates of Enterobacteriaceae in French extra-hospital practice.

Antibiotic resistance among members of the family Enterobacteriaceae was prospectively surveyed by eight French private laboratories over a 5-month period in 1999. A total of 2,599 consecutive and nonduplicate strains were collected, mainly (60.9%) from patients in the community. Most strains (82.9%) derived from urine. Escherichia coli was the predominant (73.9%) organism isolated. The overall rates of antibiotic resistance were as follows: amoxicillin, 53.4%; amoxicillin-clavulanic acid, 27.3%; ticarcillin, 44.2%; piperacillin-tazobactam, 3.2%; cephalothin, 29.2%; cefuroxime, 14.7%; cefoxitin, 11.5%; ceftazidime, 3.6%; cefotaxime, 2.8%; cefepime, 0.3%; imipenem, 0.1%; gentamicin (G), 3.8%; tobramycin (T), 5.0%; netilmicin (Nt), 3.7%; amikacin (A), 0.7%; nalidixic acid, 14.3%; ofloxacin, 10.4%; cotrimoxazole, 21.1%; nitrofurantoin, 12.7%; fosfomycin, 5.2%; tetracycline, 50.1%; and colistin, 12.5%. Beta-lactam resistance phenotypes essentially comprised penicillinase production (33.9%), overexpression of chromosomal cephalosporinase (4.6%), and synthesis of inhibitor-resistant TEM/OXA enzymes (1.5%) or extended-spectrum beta-lactamases (1.5%). Aminoglycoside resistance phenotypes consisted of GTNt (93 strains), TNtA (68 strains), GTNtA (14 strains), T (4 strains), GT (3 strains), G (1 strain), and reduced uptake/permeability (3 strains). Most of the nalidixic acid-resistant strains were resistant to ofloxacin (72.8%). Antibiotic resistance rates and phenotypes varied widely according to the bacterial group and the source of the strains. Significantly higher rates were observed in private healthcare centers than in the community, due to a higher proportion of both resistant species and resistant strains. However, multidrug-resistant isolates, including five extended-spectrum beta-lactamase-producing strains, were also recovered from the community.

The measurement of cholinesterase activities as a biomarker in chub (Leuciscus cephalus): the fish length should not be ignored.

Biomarkers are early warning systems of the exposure of aquatic organisms to pollutants. Among them, the measurement of the cholinesterase (ChE) activities in fish muscle is a biomarker of the exposure to organophosphosphates and carbamates pesticides. As such it has been used in numerous field studies both in marine and continental waters. Cyprinids (chub, Leuciscus cephalus) were sampled in river sites (France) in relatively clean and polluted areas. We performed the statistical analysis of the ChE activities and we generally observed a statistical relationship between ChE activities and fish length, the larger fish having the lower ChE activities. We concluded that the great majority of the significant differences in ChE activities between sites could be due in fact to differences in fish length between field samples. We stress the importance of taking into account the fish length whenever differences in ChE levels between field sites must be interpreted.

Multibiomarker responses in fish from the Moselle River (France).

The response of wild fish to pollutants was studied using two biomarkers in chub (Leuciscus cephalus) at five stations in the Moselle River (France) in 1998 and in 1999. The induction of cytochrome P450 1A was quantified by the ethoxyresorufin O-deethylase (EROD) activity in the liver and the level of DNA single-strand breaks was determined in erythrocytes using the comet assay. EROD activity was observed to be up to 10-fold induced in both males and females from the downstream stations in comparison to the fish from the upstream station. Levels of DNA damage did not parallel EROD induction. Chemical analyses did not clearly explain the responses of the studied biomarkers, confirming the great difficulty in relating chemical and biological information in the field. This study confirms the difficulty in assessing the biological effects of mixtures of pollutants and points out the usefulness of a large array of biomarkers.

[Antibiotic resistance of Staphylococcus aureus in urban experience: 6 month study in Aquitaine]. / Résistance aux antibiotiques de Staphylococcus aureus en pratique de ville: étude sur six mois en Aquitaine.

Antibiotic resistance of Staphylococcus aureus has been surveyed by eight city laboratories of the Aquitaine area, during a six month-period (january to june 1998). Antibiotic susceptibility testing has been performed by the disk diffusion method, and the results have been collected and analysed using the SIRscan system. After elimination of the redundant strains, a total of 747 isolates has been retained. They were mainly isolated from pus (64%) collected from patients of the community (40%) or hospitalized in 30 private clinics or nursing homes. The percentages of resistant strains (community/institutions) were: benzylpenicillin: 90% (87/92%), oxacillin: 39% (23/50%), kanamycin: 37% (22/47%); gentamicin: 13% (8/16%), tobramycin: 37% (21/47%), amikacin: 21% (13/27%); netilmicin: 6% (5/7%), erythromycin: 33% (30/35%), spiramycin: 72% (77/69%), lincomycin: 24% (17/29%), pristinamycin: 2% (1/2%), tetracycline: 17% (14/19%); pefloxacin: 40% (25/50%), fosfomycin: 9% (6/12%), rifampicin: 10% (7/13%), fusidic acid: 14% (11/15%), cotrimoxazole and vancomycin: 0%. Meticillin-susceptible strains of S. aureus were mostly sensitive to other antibiotics (< or = 6% resistant strains, except for erythromycin: 22%). Among meticillin-resistant S. aureus, heterogeneous strains with a KT phenotype, and various resistance patterns to the remaining antibiotics were predominant (61%), compared to the homogeneous strains with a KTG phenotype and multiresistant to the other antibiotics (32%). The frequencies of resistant strains were highly variable depending on the specimen, the laboratory and the health care institution, except for cotrimoxazole and vancomycin which were always active.

Two-step purification method of vitellogenin from three teleost fish species: rainbow trout (Oncorhynchus mykiss), gudgeon (Gobio gobio) and chub (Leuciscus cephalus).

A two-step purification protocol was developed to purify rainbow trout (Oncorhynchus mykiss) vitellogenin (Vtg) and was successfully applied to Vtg of chub (Leuciscus cephalus) and gudgeon (Gobio gobio). Capture and intermediate purification were performed by anion-exchange chromatography on a Resource Q column and a polishing step was performed by gel permeation chromatography on Superdex 200 column. This method is a rapid two-step purification procedure that gave a pure solution of Vtg as assessed by silver staining electrophoresis and immunochemical characterisation.

Comparative in vitro activity of cefpiramide, a new parenteral cephalosporin.

The in vitro activity of cefpiramide was compared with that of nine other cephalosporins by determining the MIC values by means of an agar dilution method for 300 representative clinical isolates of nonfastidious bacteria. Cefpiramide had a broad-spectrum of activity, similar to that of the third-generation cephalosporins. As judged by the MIC50 and the MIC90 values, cefpiramide was one of the most active cephalosporins against Pseudomonas aeruginosa (MIC50 2.9 micrograms/ml, MIC90 64 micrograms/ml), staphylococci (MIC50 1.2 micrograms/ml, MIC90 10 micrograms/ml) and Enterococcus faecalis (MIC50 13 micrograms/ml, MIC90 43 micrograms/ml). Cefpiramide had moderate activity against Enterobacteriaceae (MIC50 4 micrograms/ml, MIC90 108 micrograms/ml), comparable to that of the older second-generation cephalosporins. Ticarcillin-resistant strains of gram-negative rods were inhibited by higher concentrations of cefpiramide than ticarcillin-susceptible strains.

[In vitro action of fosfomycin combined with rifampicin, pefloxacin and imipenem on staphylococci (checkerboard method in a liquid medium)]. / Activité in vitro de la fosfomycine associée à la rifampicine, la péfloxacine et l'imipenem sur les staphylocoques (echiquier en milieu liquide).

The combined activity of fosfomycin with rifampin, pefloxacin and imipenem was investigated, by the checkerboard method performed in liquid medium, against 50 clinical isolates of staphylococci (25 Staphylococcus aureus and 25 coagulase-negative staphylococci). The combination of fosfomycin plus rifampin had an additive bacteriostatic effect and an antagonistic bactericidal effect. Fosfomycin combined with pefloxacin was found to be additive or moderately synergistic. Fosfomycin in combination with imipenem was generally highly synergistic, especially against meticillin-resistant strains; however the bactericidal effect was occasionally antagonistic.

In vitro activity of fosfomycin combined with rifampin, pefloxacin and imipenem against staphylococci: a study by the time-kill curve method.

The in vitro activity of fosfomycin alone and in combination with rifampin, pefloxacin and imipenem was studied by the time-kill method against staphylococci. Fosfomycin, pefloxacin and imipenem used at concentrations within the therapeutic range, exerted a bactericidal effect, whereas rifampin acted as a bacteriostatic drug. The combination of fosfomycin and rifampin was found to be antagonistic against rifampin-susceptible strains and indifferent for rifampin-resistant isolates. Fosfomycin combined with pefloxacin usually produced an indifferent effect. The interaction between fosfomycin and imipenem was mainly indifferent but synergism occurred with methicillin-resistant strains and antagonism was observed for one methicillin-susceptible isolate of Staphylococcus aureus.