Hypersaline water from desalinization plants causes oxidative damage in Posidonia oceanica meadows.

Posidonia oceanica is an endemic marine phanerogam of the Mediterranean Sea for that is very sensitive to the environmental changes, especially those related to human activities. The aim of this study was to evaluate the oxidative stress status of P. oceanica meadows exposed to spillage of hypersaline water from a desalination station by using biomarkers. Leaf samples of P. oceanica were obtained from 4 different points exposed to different levels of salinity water. Samples from the area with the highest salinity conditions were 75% shorter than the samples from the control area. Exposure to high salinity induced an increase in the levels of oxidative stress markers (malondialdehyde (MDA) and protein carbonyls). Interestingly, in the area with the highest salinity the activities of glutathione peroxidase, glutathione reductase and glutathione-s-transferase were significantly induced respect to the other studies areas, while catalase (CAT) and superoxide dismutase (SOD) activities were lower. In addition, salinity induced a progressive increase in non-enzymatic antioxidants (polyphenols and glutathione) and in total antioxidant capacity reaching the highest concentrations in samples directly affected by the saline discharge. In conclusion chronic exposure to high salinity induced an increase in total antioxidant capacity in P. oceanica. However, this increase was not enough to protect the plant against oxidative stress as it is evidenced by the raise in oxidative stress markers. The obtained data suggest that high salinity conditions deactivated CAT and SOD antioxidant enzymes and caused an increase in non-enzymatic antioxidants (polyphenols and glutathione) and in glutathione-related enzymes.

Sustained low prevalence of meticillin-resistant Staphylococcus aureus upon admission to hospital in The Netherlands.

The prevalence of meticillin-resistant Staphylococcus aureus (MRSA) carriage at hospital admission in The Netherlands was 0.03% in 1999-2000. The aim of the present study was to assess whether the prevalence of MRSA carriage in The Netherlands has changed over the last few years. In five Dutch hospitals, 6496 unique patients were screened for nasal S. aureus carriage at hospital admission by microbiological culture between 1 October 2005 and 7 June 2007. In total, 2036 of 6496 (31.3%) patients carried S. aureus in their nose, and seven of 6496 (0.11%) patients were nasal carriers of MRSA. Compared with 1999-2000, the prevalence of MRSA carriage in the Dutch population at hospital admission has increased more than three fold; however, this increase was not significant (P=0.06, Fisher's exact test). This prevalence is still among the lowest in the world, probably as a result of the stringent Dutch infection control policy, and the restrictive use of antibiotics in The Netherlands.

Differences in delta13C and delta15N stable isotopes in the pearly razorfish Xyrichtys novacula related to the sex, location and spawning period.

In the present study, Xyrichtys novacula (Labridae) were sampled at five locations around the islands of Ibiza and Formentera (western Mediterranean Sea). Isotopic signatures of delta13C, delta15N and the C:N ratio were analysed in relation to locality, sex and size differences. delta13C and delta15N partitioning was also studied in the reproductive spawning period. There were significant differences in the delta13C signature between localities for both sexes, but not for delta15N. Sex differences were also found with a mean +/-s.e. value of -17.38 +/- 0.06 per thousand delta13C and 8.36 +/- 0.05 per thousand delta(15)N for females and -17.17 +/- 0.07 per thousand delta13C and 8.80 +/- 0.06 per thousand delta15N for males. Increasing total length in both sexes was positively correlated with delta15N enrichment and a significant positive linear regression was established for both variables. During the reproductive spawning period, there were changes in delta13C fractioning with enrichment in postspawning females and males (with respect to prespawning and spawning periods) and delta(15)N impoverishment in postspawning females (with respect to prespawning and spawning periods). Xyrichtys novacula uses local food sources, as confirmed by delta(13)C and delta(15)N, and females and males use different food sources, thus avoiding intraspecific competition. This was confirmed by delta15N enrichment as size increased. Spawning leads to special requirements for gonad maturation, which is reflected in the isotopic signatures for both sexes.

Rapid screening of methicillin-resistant Staphylococcus aureus using PCR and chromogenic agar: a prospective study to evaluate costs and effects.

Pre-emptive isolation of suspected methicillin-resistant Staphylococcus aureus (MRSA) carriers is considered essential for controlling the spread of MRSA, but noncolonized patients will be isolated unnecessarily as a result of a delay in diagnosis of 3-5 days with conventional cultures. We determined costs per isolation day avoided, and incremental costs of rapid MRSA screening tests when added to conventional screening, but with decisions on isolation measures based on PCR results. A prospective multicentre study evaluating BD GeneOhm MRSA PCR (`IDI') (BD Diagnostics, San Diego, CA, USA), Xpert MRSA (`GeneXpert') (Cepheid, Sunnyvale, CA, USA) and chromogenic agar (MRSA-ID) (bioMérieux, Marcy-l'Etoile, France) was performed in 14 Dutch hospitals. Among 1764 patients at risk, MRSA prevalence was 3.3% (n=59). Duration of isolation was 19.7 and 16.1 h with IDI and GeneXpert, respectively, and would have been 30.0 and 76.2 h when based on chromogenic agar and conventional cultures, respectively. Negative predictive values (at a patient level) were 99.5%, 99.1% and 99.5% for IDI, GeneXpert and chromogenic agar, respectively. Numbers of isolation days were reduced by 60% and 47% with PCR-based and chromogenic agar-based screening, respectively. The cost per test was €56.22 for IDI, €69.62 for GeneXpert and €2.08 for chromogenic agar, and additional costs per extra isolation day were €26.34. Costs per isolation day avoided were €95.77 (IDI) and €125.43 (GeneXpert). PCR-based decision-making added €153.64 (IDI) and €193.84 (GeneXpert) per patient to overall costs and chromogenic testing would have saved €30.79 per patient. Rapid diagnostic testing safely reduces the number of unnecessary isolation days, but only chromogenic screening, and not PCR-based screening, can be considered as cost saving.

Barminomycin, a model for the development of new anthracyclines.

Barminomycin is a member of the anthracycline class of anticancer agents and was originally discovered as a pink/red complex with DNA and RNA and named SN-07. The chromophore was subsequently separated from the nucleic acids by nuclease digestion and contained the four-membered anthraquinone ring system characteristic of anthracyclines, but with an unusual eight membered ring that contained a carbinolamine which readily interconverted to an imine. The imine form is analogous to the formaldehyde-activated form of other anthracyclines such as doxorubicin. The imine form confers exceptional activity to barminomycin which is 1,000-fold more cytotoxic than doxorubicin. Barminomycin rapidly forms adducts with DNA, reacting with the exocyclic amino group of guanine residues and with high selectivity for 5'-GC-3' sequences. The coupling to DNA appears to be identical to the N-C-N aminal linkage formed between doxorubicin and DNA where the carbon derives from formaldehyde for doxorubicin-DNA adducts, whereas this "activated carbon" is an inherent component of the imine group in the eight membered ring of barminomycin. Although the linkage of both drugs to DNA appears to be identical, barminomycin-DNA complexes are essentially irreversible compared to the labile doxorubicin-DNA adducts which have an in vitro (purified DNA) half-life of 25 h at 37 degrees C. A 3D model of the barminomycin-DNA complex has been defined from 307 NOE distance constraints. The enhanced stability of barminomycin-DNA adducts appears to be due primarily to protection of the aminal linkage from hydrolysis and this has provided insight into the design of new anthracycline derivatives with enhanced stability and activity. Strategies for harnessing the extreme reactivity and activity of barminomycin are also presented.

Muscle and scale isotopic offset of three fish species in the Mediterranean Sea: Dentex dentex, Argyrosomus regius and Xyrichtys novacula.

Diet information of organisms was traditionally acquired by the use of lethal techniques (gut content or muscle delta(13)C and delta(15)N isotopic ratios). An increase in the number of isotopic ratio studies and the vulnerability of some species have led to increased use of non-lethal methodologies for conservation purposes. In the present work we have compared the delta(13)C and delta(15)N isotopic signals of muscle and scales of three different fish species in order to test fish-scale sampling as a non-lethal technique in fish trophodynamics. A positive correlation was found between muscle and scales in Argyrosomus regius and Xyrichtys novacula, while Dentex dentex showed no correlation due to the small length range of this species. The isotopic offset (Delta(13)C and Delta(15)N) between muscle and scales was significantly different among species (analysis of variance (ANOVA), p < 0.001) with Tukey's post-hoc HSD indicating a tissue offset difference (p < 0.001) for both stable isotopes in all species, except for Delta(13)C between A. regius and X. novacula and for Delta(15)N between D. dentex and X. novacula (p > 0.05). Mean delta(13)C and delta(15)N values between species showed significant differences (paired t-test, p < 0.01) between muscle and scale with an enrichment for delta(13)C and a depletion for delta(15)N. Spatial differences were found in the Delta(13)C of X. novacula between the studied locations, while no differences were found for Delta(15)N, indicating that non-geographical differences should be considered in the application of scales instead of muscle for (15)N, while for (13)C differences in the geographical isotopic offset should be considered.

Stable isotopes and metal contamination in caged marine mussel Mytilus galloprovincialis.

Metal concentrations and isotopic composition were measured in different tissues of the mussel Mytilus galloprovincialis in waters of the Balearic Islands (Western Mediterranean) in order to assess pollution levels. The isotopic composition was correlated with lead, cadmium, selenium and nickel obtained from the digestive gland and foot of the mussels. Significant negative correlations were found between cadmium, selenium and zinc and the mussel foot, mainly for (13)C. Significant correlations were also found between lead and cadmium and the digestive gland. Pearson correlations indicated that the (13)C isotopic signal in foot is a good proxy for the concentration of metals such as lead, cadmium, selenium and zinc. Similarly, (15)N isotopic signatures in the digestive gland reflected the lead and cadmium concentration.

Assessment of environmental pollution at Balearic Islands applying oxidative stress biomarkers in the mussel Mytilus galloprovincialis.

The antioxidant enzyme response of the mussel Mytilus galloprovincialis to different degree of pollution was investigated. Antioxidant enzyme activities - catalase (CAT), glutathione peroxidase (GSH-PX), glutathione reductase (GR), superoxide dismutase (SOD) - and malondialdehyde (MDA) concentration were measured in gills and digestive glands of mussels. Mussels from the same origin were transplanted along the Balearic coastal waters in eight stations characterized by a different degree of contamination and human impacts. Antioxidant enzyme activities showed an adaptive response to increase the activities in the more polluted areas. CAT, GR and SOD in gills and CAT and GR in digestive gland presented significant differences between polluted and non-polluted stations. No significant differences were observed in MDA concentration indicating that the antioxidant response is capable to avoid the lipid peroxidation. The use of biomarkers such as CAT and GR in gills and digestive glands of the mussel M. galloprovincialis is a good tool to categorize differences between polluted and non-polluted areas.

Organic compounds temporal trends at some invertebrate species from the Balearics, Western Mediterranean.

Concentrations of persistent organic pollutants (POPs) such as hexachlorobenzene (HCB), dichlore diphenyl trichloretane (DDT), polychlorinated biphenyls (PCBs), and gamma-hexachlorocyclohexane (gamma-HCH or lindane) were determined in tissue of marine benthic invertebrates such as Mytilus galloprovincialis, Chamelea gallina, Venus verrucosa, Lithophaga lithophaga and Paracentrotus lividus. Species were selected due to their habitat, trophic level, feeding behaviour and their consumption. Invertebrate species were systematically sampled from December 1996 to December 2005 from several sites along the Balearic Islands. The highest concentrations of PCBs (785ng/g lipid) were found in M. galloprovincialis while the lowest concentrations were found in the sea-urchin P. lividus (193ng/g lipid). Among the 7 PCB quantified congeners the higher values are mainly obtained for CB138 and CB153. All bivalves presented higher PCBs contents than the sea-urchin P. lividus are possibly linked with the bioaccumulation process of POPs throughout the food web and to differential detoxifying mechanisms. The concentration of SigmaDDT exceeds that of HCB and gamma-HCH at all species and sampling stations. DDT concentrations ranged from 0.4ng/g ww at the bivalve C. gallina in 2002, to values of 15.8ng/g ww at the bivalve L. lithophaga in 1998. The values obtained for the organic compounds (HCH, HCB, PCBs, DDT) depend upon the place and year of sampling and are compared to values found by other authors for the mussel M. galloprovincialis in other Mediterranean areas. gamma-HCH and HCB were found in lower concentrations than the other POPs.

Methicillin-resistant staphylococci isolated from animals.

Staphylococci isolated from animals (n=311) were screened for methicillin resistance by oxacillin agar screening. Oxacillin-resistant strains were tested for the presence of the mecA gene by PCR. Isolates were identified by standard techniques and 16S rDNA analysis, and their antimicrobial susceptibilities were tested using an agar diffusion method. MecA-positive strains were further analyzed using pulsed-field gel electrophoresis (PFGE). From 11 multidrug-resistant staphylococci, 6 were mecA-positive: 2 methicillin-resistant Staphylococcus aureus (MRSA) and 4 Staphylococcus haemolyticus. Screening of 300 staphylococci (100 S. aureus, 100 S. intermedius and 100 coagulase-negative staphylococci (CNS)) randomly chosen from the strain collection of the Veterinary Microbiological Diagnostic Center yielded five oxacillin-resistant coagulase-negative staphylococci, four of which were mecA-positive. PFGE showed that all mecA-positive staphylococci isolated from animals had distinct patterns. However, one MRSA isolated from a flank fistula of a dog showed homology to a human epidemic MRSA cluster, suggesting that transfer of MRSA between humans and dogs might occur.

Evaluation of genetic determinants involved in beta-lactam- and multiresistance in a surgical ICU.

Antibiotic resistance is a major and well-known problem in intensive care units (ICUs) world-wide and previously susceptible isolates become resistant through the acquisition of resistance determinants from other bacteria or the development of mutations, as is the case in beta-lactam resistance. We evaluated the presence of resistance determinants involved in beta-lactam resistance and multi-resistance in order to establish the contribution of horizontal gene transfer to the spread of resistance in a surgical ICU during an antibiotic rotation study. Pseudomonas aeruginosa and Enterobacteriaceae isolates were selected and iso-electric focusing (IEF), DNA-typing methods such as specific beta-lactamase and specific integron PCRs were performed to determine the presence of beta-lactamases. The PCRs specific for IMP-1, OXA-1, and VIM-type beta-lactamases performed on the selected P. aeruginosa and Enterobacteriaceae isolates with MICs for cephalosporins >1 mg/l did not demonstrate any of these beta-lactamases. IEF for 14 pseudomonads, representing 7 genotypes from 9 patients, showed a beta-lactamase with a pI larger than 8.5 in 13 of the isolates. The integrase PCR was positive for only five isolates from three patients and conserved segment PCR showed integrons of variable sizes (700, 900, 1,400 and 1,500 bp). Each patient had its own integron types. It can be concluded that integrons and associated resistance determinants played only a minor role in the surgical ICU and beta-lactam resistance among P. aeruginosa isolates was most likely due to the derepression of its AmpC gene.

False-positive results and contamination in nucleic acid amplification assays: suggestions for a prevent and destroy strategy.

Contamination of samples with DNA is still a major problem in microbiology laboratories, despite the wide acceptance of PCR and other amplification techniques for the detection of frequently low amounts of target DNA. This review focuses on the implications of contamination in the diagnosis and research of infectious diseases, possible sources of contaminants, strategies for prevention and destruction, and quality control. Contamination of samples in diagnostic PCR can have far-reaching consequences for patients, as illustrated by several examples in this review. Furthermore, it appears that the (sometimes very unexpected) sources of contaminants are diverse (including water, reagents, disposables, sample carry over, and amplicon), and contaminants can also be introduced by unrelated activities in neighboring laboratories. Therefore, lack of communication between researchers using the same laboratory space can be considered a risk factor. Only a very limited number of multicenter quality control studies have been published so far, but these showed false-positive rates of 9-57%. The overall conclusion is that although nucleic acid amplification assays are basically useful both in research and in the clinic, their accuracy depends on awareness of risk factors and the proper use of procedures for the prevention of nucleic acid contamination. The discussion of prevention and destruction strategies included in this review may serve as a guide to help improve laboratory practices and reduce the number of false-positive amplification results.

[Methicillin resistant Staphylococcus aureus (MRSA) infection in a dog in the Netherlands]. / Een meticilline-resistente Staphylococcus aureus (MRSA)-infectie bij een hond in Nederland.

In the Netherlands, methicillin resistant Staphylococcus aureus (MRSA) are regularly isolated from humans. We present the first isolation of MRSA from animal origin in the Netherlands. A coagulase positive staphylococcus was cultured from an infected wound in a Dutch dog that recently underwent surgery abroad. The staphylococcus was resistant to methicillin, ampicillin, amoxycillin + clavulanic acid, cephalexin, erythromycin, lincomycin, tetracycline, gentamicin and enrofloxacin. It was identified as S. aureus by fermentation of mannitol and Martineau-PCR. The presence of mecA was confirmed by PCR.

Presence of integron-associated resistance in the community is widespread and contributes to multidrug resistance in the hospital.

Integrons are strongly associated with the multidrug resistance seen in gram-negative bacilli in the hospital environment. No data, however, are available on their prevalence in the community. This study is the first to show that integrons are widespread in Enterobacteriaceae in the community and that integron-associated resistance genes in the community constitute a substantial reservoir for multidrug resistance in the hospital.

Novel gene cassettes and integrons.

An increase in multiresistant Enterobacteriaceae was observed at one of the departments of the University Medical Center Utrecht. Nine different integrons and 17 gene cassettes were found, including the new gene cassette aadA8. This cassette was highly related to aadA3 and aadA2. In addition, an unknown promoter sequence was found for two integrons.

The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body.

The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

Control of nosocomial multiresistant Enterobacteriaceae using a temporary restrictive antibiotic agent policy.

An observational study on the epidemiology of multiresistant Enterobacteriaceae was conducted in the neurology and neurosurgery wards of a university hospital to determine the impact of hospital hygiene measures and an additional temporary restrictive antibiotic agent policy on the sudden rise in incidence of these bacteria. The incidence and prevalence of patients with multiresistant Enterobacteriaceae were assessed, and patient isolates were typed phenotypically and by random amplified polymorphic DNA analysis. All hospital hygiene measures implemented were recorded, and the influence of the restrictive policy on antibiotic use was analyzed. This policy consisted of a prior authorization requirement and the withdrawal of all antibiotics with a possible selective pressure on multiresistant strains (gentamicin, tobramycin, quinolones, cotrimoxazole, broad-spectrum penicillins, and cephalosporins). This ban left only carbapenems and amikacin for treatment. Typing showed that 17 of the 61 (28%) patients involved were infected or colonized with a single multiresistant strain of Klebsiella oxytoca, for which an environmental source was identified. The isolates recovered from the other patients comprised eight different species, and subsequent genotyping yielded a great variety of strains. The increased incidence could not be controlled with hospital hygiene measures alone. Only after implementation of the restrictive antibiotic policy did the epidemic strain vanish and the endemic incidence of multiresistant Enterobacteriaceae decrease to <50% of the level before intervention. In the years since, the incidence has remained at this low level, and the antibiotic costs have decreased to a level lower than before intervention.

Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species.

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.