Phylogeny and systematics of demospongiae in light of new small-subunit ribosomal DNA (18S) sequences.

The most diverse and species-rich class of the phylum Porifera is Demospongiae. In recent years, the systematics of this clade, which contains more than 7000 species, has developed rapidly in light of new studies combining molecular and morphological observations. We add more than 500 new, nearly complete 18S sequences (an increase of more than 200%) in an attempt to further enhance understanding of the phylogeny of Demospongiae. Our study specifically targets representation of type species and genera that have never been sampled for any molecular data in an effort to accelerate progress in classifying this diverse lineage. Our analyses recover four highly supported subclasses of Demospongiae: Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha. Within Keratosa, neither Dendroceratida, nor its two families, Darwinellidae and Dictyodendrillidae, are monophyletic and Dictyoceratida is divided into two lineages, one predominantly composed of Dysideidae and the second containing the remaining families (Irciniidae, Spongiidae, Thorectidae, and Verticillitidae). Within Myxospongiae, we find Chondrosida to be paraphyletic with respect to the Verongida. We amend the latter to include species of the genus Chondrosia and erect a new order Chondrillida to contain remaining taxa from Chondrosida, which we now discard. Even with increased taxon sampling of Haploscleromorpha, our analyses are consistent with previous studies; however, Haliclona species are interspersed in even more clades. Haploscleromorpha contains five highly supported clades, each more diverse than previously recognized, and current families are mostly polyphyletic. In addition, we reassign Janulum spinispiculum to Haploscleromorpha and resurrect Reniera filholi as Janulum filholi comb. nov. Within the large clade Heteroscleromorpha, we confirmed 12 recently identified clades based on alternative data, as well as a sister-group relationship between the freshwater Spongillida and the family Vetulinidae. We transfer Stylissa flabelliformis to the genus Scopalina within the family Scopalinidae, which is of uncertain position. Our analyses uncover a large, strongly supported clade containing all heteroscleromorphs other than Spongillida, Vetulinidae, and Scopalinidae. Within this clade, there is a major division separating Axinellidae, Biemnida, Tetractinellida, Bubaridae, Stelligeridae, Raspailiidae, and some species of Petromica, Topsentia, and Axinyssa from Agelasida, Polymastiidae, Placospongiidae, Clionaidae, Spirastrellidae, Tethyidae, Poecilosclerida, Halichondriidae, Suberitidae, and Trachycladus. Among numerous results: (1) Spirophorina and its family Tetillidae are paraphyletic with respect to a strongly supported Astrophorina within Tetractinellida; (2) Agelasida is the earliest diverging lineage within the second clade listed above; and (3) Merlia and Desmacella appear to be the earliest diverging lineages of Poecilosclerida.

Transformation of a cranial fusiform aneurysm into a pseudotumoral-like mass prior to spontaneous occlusion and regression.

This report documents the evolution of a non-ruptured cranial fusiform aneurysm that underwent both spontaneous occlusion and regression. During this process, unique images of the aneurysm as a pseudotumoral-like mass were obtained. The pseudotumoral-like mass most likely reflected inflammation and secondary neovascularization within the aneurysm, supporting the theory that spontaneous aneurysmal healing involves an inflammatory process.

Diversity of bacteria in the marine sponge Aplysina fulva in Brazilian coastal waters.

Microorganisms can account for up to 60% of the fresh weight of marine sponges. Marine sponges have been hypothesized to serve as accumulation spots of particular microbial communities, but it is unknown to what extent these communities are directed by the organism or the site or occur randomly. To address this question, we assessed the composition of specific bacterial communities associated with Aplysina fulva, one of the prevalent sponge species inhabiting Brazilian waters. Specimens of A. fulva and surrounding seawater were collected in triplicate in shallow water at two sites, Caboclo Island and Tartaruga beach, Búzios, Brazil. Total community DNA was extracted from the samples using "direct" and "indirect" approaches. 16S rRNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the total bacterial community and of specific bacterial groups--Pseudomonas and Actinobacteria--revealed that the structure of these assemblages in A. fulva differed drastically from that observed in seawater. The DNA extraction methodology and sampling site were determinative for the composition of actinobacterial communities in A. fulva. However, no such effects could be gleaned from total bacterial and Pseudomonas PCR-DGGE profiles. Bacterial 16S rRNA gene clone libraries constructed from directly and indirectly extracted DNA did not differ significantly with respect to diversity and composition. Altogether, the libraries encompassed 15 bacterial phyla and the candidate division TM7. Clone sequences affiliated with the Cyanobacteria, Chloroflexi, Gamma- and Alphaproteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria were, in this order, most abundant. The bacterial communities associated with the A. fulva specimens were distinct and differed from those described in studies of sponge-associated microbiota performed with other sponge species.

Hungarian hospital antibiotic consumption at the regional level, 1996-2005.

BACKGROUND: Regional variations in antibiotic consumption in outpatients have been reported previously, but nothing is as yet known about the regional distribution of antibiotic consumption in the hospital sector in Hungary. This study was designed to explore regional variations and investigate determinants of antibiotic consumption in hospital care in Hungary. MATERIALS AND METHODS: Regional distribution-based antibiotic sales data were obtained for a 10-year period (1996-2005) for the 20 Hungarian counties. Systemic antibacterial use (Anatomical Therapeutic Chemical code: J01) was expressed as the number of defined daily doses (DDD) per 100 patient-days. The multiple linear regression model was applied to investigate the determinants of regional differences in hospital antibiotic consumption. Independent variables related to health care access, utilization of hospital resources, doctors' workload, type of hospital care provided, and patient's characteristics and infections were considered as possible determinants, and data on these variables were obtained for 2 years (2004, 2005). We also tested the association between hospital and ambulatory care antibiotic consumption in Hungarian regions using the Pearson correlation test. RESULTS: For each year during the 1996-2005 study period, there were large and stable variations in total hospital antibiotic consumption (e.g., min-max(1996): 16.0-28.2; min-max(2005): 15.2-32.2 DDD per 100 patient-days) depending on the region. In the two developed models (Model 1 and Model 2), the number of reported infections accounted for 53% of the observed regional variations in hospital antibiotic consumption (Model 1), and the number of reported infections together with the case-mix index were responsible for 61% (Model 2) . Total antibiotic consumption in hospitals showed a positive correlation (R = 0.71, p = 0.002) with total antibiotic consumption in ambulatory care. CONCLUSION: The case-mix index and the number of reported infections explained some of the observed regional variations. However, the moderate value of the models in explaining these regional variations suggest that determinants which could not be explored in this preliminary study may also contribute to regional differences. Future studies should aim at collecting data for each individual hospital as well as data on possible determinants for hospital antibiotic consumption.

Results of a local antibiotic management program on antibiotic use in a tertiary intensive care unit in Hungary.

BACKGROUND: Massive antibiotic use in intensive care units (ICU) is associated with increased microbial resistance. Therefore avoiding unneccesary antibiotic usage is essential. To achieve a more considered antibiotic prescribing practice, a new antibiotic policy was implemented at our ICU. In this paper, we evaluated the impact of this intervention, and described the aetiology and incidence of blood stream infections and selected antibiotic-resistant pathogens. MATERIALS AND METHODS: In November 2002, a local antibiotic management program (LAMP) was implemented. This included a new infectious diseases specialist consultation service and restricted authorisation to prescribe antibiotics. The effect on ward-level antibiotic use was examined by segmented regression analysis. Patient, ICU and microbiology data were also recorded and compared before and after policy implementation. RESULTS: The patient populations and the subsequent mortality rate were comparable before and after the implementation of the policy. Total antibiotic consumption was markedly reduced from 162.9 to 101.3 defined daily dose (DDD) per 100 patients, and per day (DDD per 100 patient-days). This was mainly accounted for a reduction in the use of quinolones, aminoglycosides, glycopeptides, metronidazol, carbepenems and third generation cephalosporins. CONCLUSION: This study has confirmed that establishing a targeted LAMP, based on close co-operation between intensive care physicians and infectious disease specialists together with a restricted prescribing authority, can reduce the use of antibiotics.

An 8-year evaluation of antibiotic consumption and antibiotic resistance among Streptococcus pneumoniae from in- and out-patients in Szeged, Hungary.

At the beginning of the 1990s, the prevalence of penicillin resistance of Streptococcus pneumoniae strains in Hungary was found to be extremely high (up to 58% non-susceptible) in some studies, while in other publications the percentage of penicillin highly resistant strains was 0-2%. To see whether this was due to differences in methodology or the composition of the patient population studied, a retrospective evaluation was carried out of the penicillin, amoxicillin, ceftriaxone and macrolide resistance of all S. pneumoniae strains isolated from in- and outpatients in our laboratory between 1998 and 2005. Of the 2670 S. pneumoniae isolates only 5.58% was found to exhibit high-level resistance to penicillin, while resistance to amoxicillin, ceftriaxone and erythromycin was 2.62%, 1.12% and 42.06%, respectively. During this period 6 (3.8%) of 155 S. pneumoniae strains isolated from invasive samples displayed high-level resistance to penicillin. Earlier surveillance data on penicillin resistance of S. pneumoniae may have been biased by the age groups affected by the infection, by whether the strain was isolated from an out-patient or an in-patient, and by whether the isolates were obtained from invasive samples. Our 8-year study using the NCCLS/CLSI methodology consequently revealed a low prevalence of high-level resistance to penicillin in S. pneumoniae strains obtained both from adults and children.

The relationship between serotypes and PFGE genotypes in isolates of Streptococcus pneumoniae from Hungary.

The relatedness of 112 penicillin-non-susceptible isolates of Streptococcus pneumoniae from Hungary was determined by pulsed-field gel electrophoresis (PFGE), serotyping and antibiotic susceptibility tests. The differences in PFGE patterns closely mirrored the changes in resistance. Some genotypes comprised multiple serotypes, and the genetic diversity among certain serotypes was considerable. Generally, serotyping alone was insufficient for epidemiological mapping of pneumococcal isolates. There was considerable serotype diversity, but the five most frequent international serotypes (6, 9, 14, 23, 19) were the most prevalent. In addition, the presence of some well-defined resistant international pneumococcal clones in the Hungarian population was identified.

Arenosclerins A-C and haliclonacyclamine E, new tetracyclic alkaloids from a Brazilian endemic Haplosclerid sponge Arenosclera brasiliensis.

Arenosclerins A (2), B (3), and C (4), as well as haliclonacyclamine E (1), are new tetracyclic alkylpiperidine alkaloids isolated from a new species of marine sponge belonging to the order Haplosclerida, Arenosclera brasiliensis, a species endemic to the southeastern Brazilian coast. The alkaloids were isolated as their hydrochloride salts and identified by analysis of spectroscopic data. Data obtained from (1)H-(1)H COSY, HMBC, and HSQC-TOCSY NMR experiments allowed complete assignment of the (1)H and (13)C resonances, and analysis of the NOESY and ROESY spectra showed that the only differences between 2, 3, and 4 were the relative stereochemistries of the bispiperidine ring system. Arenosclerins A-C are the first haliclonacyclamine/halicyclamine-related alkaloids with a hydroxy group in the bridging alkyl chain.

Characterization of 2'-deoxyadenosine adducts derived from 4-oxo-2-nonenal, a novel product of lipid peroxidation.

Analysis of the reaction between 2'-deoxyadenosine and 4-oxo-2-nonenal by liquid chromatography/mass spectrometry revealed the presence of three major products (adducts A(1), A(2), and B). Adducts A(1) and A(2) were isomeric; they interconverted at room temperature, and they each readily dehydrated to form adduct B. The mass spectral characteristics of adduct B obtained by collision-induced dissociation coupled with multiple tandem mass spectrometry were consistent with those expected for a substituted etheno adduct. The structure of adduct B was shown by NMR spectroscopy to be consistent with the substituted etheno-2'-deoxyadenosine adduct 1' '-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]heptane-2' '-one. Unequivocal proof of structure came from the reaction of adducts A(1) and A(2) (precursors of adduct B) with sodium borohydride. Adducts A(1) and A(2) each formed the same reduction product, which contained eight additional hydrogen atoms. The mass spectral characteristics of this reduction product established that the exocyclic amino group (N(6)) of 2'-deoxyadenosine was attached to C-1 of the 4-oxo-2-nonenal. The reaction of 4-oxo-2-nonenal with calf thymus DNA was also shown to result in the formation of substituted ethano adducts A(1) and A(2) and substituted etheno adduct B. Adduct B was formed in amounts almost 2 orders of magnitude greater than those of adducts A(1) and A(2). This was in keeping with the observed stability of the adducts. The study presented here has provided additional evidence which shows that 4-oxo-2-nonenal reacts efficiently with DNA to form substituted etheno adducts.

Flavopiridol binds to duplex DNA.

Flavopiridol, the first potent cyclin-dependent kinase inhibitor to enter clinical trials, was recently found to be cytotoxic to noncycling cells. The present studies were performed to examine the hypothesis that flavopiridol, like several other antineoplastic agents that kill noncycling cells, might also interact with DNA. Consistent with this possibility, treatment of A549 human lung cancer cells with clinically achievable concentrations of flavopiridol resulted in rapid elevations of the DNA damage-responsive protein p53. In further studies, the binding of flavopiridol to DNA was examined in vitro by four independent techniques. Absorption spectroscopy revealed that addition of DNA to aqueous flavopiridol solutions resulted in a red shift of the flavopiridol lambda(max) from 311 to 344 nm, demonstrating an isosbestic point typical of changes seen with DNA-binding compounds. Reverse-phase high-performance liquid chromatography demonstrated that flavopiridol binds to genomic DNA to a similar extent as ethidium bromide and Hoechst 33258. Nuclear magnetic resonance spectroscopy revealed that DNA caused extreme broadening of flavopiridol 1H nuclear magnetic resonance signals that could be reversed by addition of ethidium bromide or by DNA melting, suggesting that flavopiridol binds to (and likely intercalates into) duplex DNA. Equilibrium dialysis demonstrated that the equilibrium dissociation constant of the flavopiridol-DNA complex (5.4+/-3.4 x 10(-4) M) was in the same range observed for binding of the intercalators doxorubicin and pyrazoloacridine to DNA. Molecular modeling confirmed the feasibility of flavopiridol intercalation into DNA and analysis of the effects of flavopiridol in the National Cancer Institute tumor cell line panel using the COMPARE algorithm demonstrated that flavopiridol most closely resembles cytotoxic antineoplastic intercalators. Collectively, these data suggest that DNA might be a second target of flavopiridol, providing a potential explanation for the ability of this agent to kill noncycling cancer cells.

Novel polycyclic guanidine alkaloids from two marine sponges of the genus Monanchora.

Two marine sponges of the genus Monanchora (Poecilosclerida, Crambeidae) have been found to contain new polycyclic guanidine alkaloids bearing the (5,6,8b)-triazaperhydroacenaphthylene skeleton. Their structures have been determined by detailed spectroscopic analysis. Dehydrobatzelladine C (1) has been isolated from M. arbuscula and crambescidins 359 (2) and 431 (3) from M. unguiculata. The chemotaxonomic implications of these findings are discussed.

Metabolism and excretion of [(14)C]celecoxib in healthy male volunteers.

We determined the disposition of a single 300-mg dose of [(14)C]celecoxib in eight healthy male subjects. The [(14)C]celecoxib was administered as a fine suspension reconstituted in 80 ml of an apple juice/Tween 80/ethanol mixture. Blood and saliva samples were collected at selected time intervals after dosing. All urine and feces were collected on the 10 consecutive days after dose administration. Radioactivity in each sample was determined by liquid scintillation counting or complete oxidation and liquid scintillation counting. Metabolic profiles in plasma, urine, and feces were obtained by HPLC, and metabolites were identified by mass spectrometry and NMR. [(14)C]Celecoxib was well absorbed, reaching peak plasma concentrations within 2 h of dosing. [(14)C]Celecoxib was extensively metabolized, with only 2.56% of the radioactive dose excreted as celecoxib in either urine or feces. The total percentage of administered radioactive dose recovered was 84.8 +/- 4.9%, with 27.1 +/- 2.2% in the urine and 57.6 +/- 7.3% in the feces. The oxidative metabolism of celecoxib involved hydroxylation of celecoxib at the methyl moiety followed by further oxidation of the hydroxyl group to form a carboxylic acid metabolite. The carboxylic acid metabolite of celecoxib was conjugated with glucuronide to form the 1-O-glucuronide. The percentages of the dose excreted in the feces as celecoxib and the carboxylic acid metabolite were 2.56 +/- 1.09 and 54.4 +/- 6.8%, respectively. The majority of the dose excreted in the urine was the carboxylic acid metabolite (18.8 +/- 2.1%); only a small amount was excreted as the acyl glucuronide (1.48 +/- 0.15%).

Natural deoxynivalenol (DON) contamination of wheat samples grown in 1998 as determined by high-performance liquid chromatography.

A high-performance liquid chromatography--diode array detection (HPLC-DAD) method was developed for determining the deoxynivalenol (DON) content of wheat and other cereals. The samples were extracted with a mixture of acetonitrile and water (84 + 16). Part of the extract was evaporated and purified on Florisil and activated charcoal columns. HPLC separation was performed on a C18 column, using acetonitrile-water (8 + 92) as eluent. Diode array detection (DAD) was performed at 218 and 236 nm, by determination of the UV spectrum. Quantitative analysis was carried out by the external standard method, using the UV spectrum obtained by DAD for confirmation. The recovery rate of DON was 75 +/- 3.1% and the detection limit was 0.05 mg/kg DON. Using this method, the DON content of 99 feeding wheat samples grown in the northeastern part of Hungary in 1998 was determined. Eighty-eight percent of the samples originating from three counties contained 0.94 mg/kg DON on the average. The highest individual value was 4.3 mg/kg. DON contamination of wheat was of higher prevalence (100%) and severity (0.27-4.3 mg/kg) in the southeastern county of Békés than in Szabolcs county located in the northeastern part of Hungary (ratio of positive samples: 82%; DON concentration: 0.05-1.3 mg/kg). The higher than usual DON contamination of feeding wheat can be explained by the rainy summer weather. DON contamination of feeding wheat poses a major risk to the production and animal health status of pig herds.

Reusability of immunoaffinity columns for determination of fumonisins in maize.

Eighteen maize samples were assayed for fumonisin B1 (FB1) and B2 content by immunoaffinity column coupled with high performance liquid chromatography (HPLC). The FumoniTest columns were used once for the isolation of fumonisins (single-use column method). In the second part of the assay the columns were regenerated. After elution with methanol, PBS solution was left on the column for one day at room temperature to regenerate the columns (regenerated column method). The efficiency of columns regenerated twice was tested by determining FB, recovery and the reproducibility of the determinations. The recovery rate of FB1 proved to be 82% by the single-use column method (RSD: 5.7%) and 82.6% (RSD: 5.6 %) by the regenerated column method; 500-8,000 ng FB1 loaded onto the columns did not affect column performances. Nearly identical values were obtained when the FB1 content of fumonisin-containing maize samples was determined by both methods. The results indicate that the FumoniTest columns can be regenerated by the method applied at least twice without decrease in column performance. The fumonisin affinity, capacity and specificity of the regenerated columns were not changed. Thus, columns regenerated in this way can be used for determining the fumonisin content of maize samples at least three times.

Investigation of the presence of different broad-spectrum beta-lactamases among clinical isolates of Enterobacteriacae.

Chromosomal or plasmid-encoded beta-lactamases are the most frequent causes of resistance to broad-spectrum beta-lactam antibiotics in clinical isolates of Gram-negative bacteria. Different screening methods can be used for their detection during routine laboratory work, while molecular biological methods may help in the detection of the genetic background of the phenotypic resistance. Clinical isolates of Klebsiella pneumoniae (170) and Enterobacter cloaceae (82) were obtained from different parts of Hungary, whereas those of Serratia marcescens (15) were isolated in our Department from a nosocomial outbreak. Disk diffusion and the Etest were used to screen inducible Class C beta-lactamase and plasmid-mediated extended spectrum beta-lactamases (ESBLs) among clinical isolates of Enterobacteriaceae. Single-strand conformation polymorphism (SSCP) analysis of the PCR products obtained after using SHV-specific primers revealed the presence of SHV-2 and SHV-5 ESBL among 170 K. pneumoniae strains in 12 and 3 cases, respectively. The results of the screening methods and the PCR-SSCP analysis suggested that 14 of the 15 S. marcescens strains not only produced the Class C, inducible chromosomal beta-lactamase, but also acquired a plasmid-mediated SHV-2-type ESBL. One strain isolated from the environment during the outbreak was genetically related to the other isolates, as demonstrated by the different typing methods, but it did not produce ESBL. The in vivo transfer of SHV-2 gene was assumed from an SHV-2 positive K. pneumoniae strain present in the same ward, in the same patient and at the same time. A very high prevalence of the stable derepressed mutants of E. cloaceae was confirmed among the Hungarian isolates. Seventy seven per cent of the strains produced high amounts of beta-lactamase without induction being responsible for their resistance to third-generation cephalosporins. Nineteen per cent of the strains were inducible when cefoxitin or imipenem was used, as confirmed by direct measurement of the MICs with the Etest.