Evolution of fluoroquinolone-resistant Escherichia coli in the gut after ciprofloxacin treatment.

BACKGROUND: Three healthy volunteers carried similar quinolone-resistant E. coli (QREC) (pulsed field gel electrophoresis profiles) in their gut before and after 14 days ciprofloxacin treatment. Given the intensity of the selective pressure and the mutagenic properties of quinolones, we determined whether these strains had evolved at the phenotypic and/or genomic levels. MATERIAL AND METHODS: Commensal QREC from before day-0 (D0), and a month after 14 days of ciprofloxacin (D42) were compared in 3 volunteers. Growth experiments were performed; acetate levels, mutation frequencies, quinolone MICs and antibiotic tolerance were measured at D0 and D42. Genomes were sequenced and single nucleotide polymorphisms (SNPs) between D0 and D42 were analyzed using DiscoSNP and breseq methods. Cytoplasmic proteins were extracted, HPLC performed and proteins identified using X!tandem software; abundances were measured by mass spectrometry using the Spectral Counting (SC) and eXtraction Ion Chromatograms (XIC) integration methods. RESULTS: No difference was found in MICs, growth characteristics, acetate concentrations, mutation frequencies, tolerance profiles, phylogroups, O-and H-types, fimH alleles and sequence types between D0 and D42. No SNP variation was evidenced between D0 and D42 isolates for 2/3 subjects; 2 SNP variations were evidenced in one. At the protein level, very few significant protein abundance differences were identified between D0 and D42. CONCLUSION: No fitness, tolerance, metabolic or genomic evolution of commensal QREC was observed overtime, despite massive exposure to ciprofloxacin in the gut. The three strains behaved as if they had been unaffected by ciprofloxacin, suggesting that gut may act as a sanctuary where bacteria would be protected from the effect of antibiotics and survive without any detrimental effect of stress.

The decoupling between genetic structure and metabolic phenotypes in Escherichia coli leads to continuous phenotypic diversity.

To assess the extent of intra-species diversity and the links between phylogeny, lifestyle (habitat and pathogenicity) and phenotype, we assayed the growth yield on 95 carbon sources of 168 Escherichia strains. We also correlated the growth capacities of 14 E. coli strains with the presence/absence of enzyme-coding genes. Globally, we found that the genetic distance, based on multilocus sequence typing data, was a weak indicator of the metabolic phenotypic distance. Besides, lifestyle and phylogroup had almost no impact on the growth yield of non-Shigella E. coli strains. In these strains, the presence/absence of the metabolic pathways, which was linked to the phylogeny, explained most of the growth capacities. However, few discrepancies blurred the link between metabolic phenotypic distance and metabolic pathway distance. This study shows that a prokaryotic species structured into well-defined genetic and lifestyle groups can yet exhibit continuous phenotypic diversity, possibly caused by gene regulatory effects.

Identification of patients with impaired hepatic drug metabolism using a limited sampling procedure for estimation of phenazone (antipyrine) pharmacokinetic parameters.

Phenazone (antipyrine) 1g was given by short intravenous infusion to 62 study participants (10 healthy drug-free volunteers and 52 patients with chronic liver disease). A Bayesian approach was developed to determine the individual pharmacokinetic parameters of phenazone. Statistical characteristics of the population pharmacokinetic parameters were first evaluated for 30 patients. When combined with 1 plasma drug concentration from members of the second group, these led to a Bayesian estimation of individual pharmacokinetic parameters for the remaining 32 individuals. Total clearance computed by Bayesian estimation was compared with maximal likelihood estimation of this parameter, the classical procedure. No statistically significant differences were found. Performance of the developed methodology was evaluated by computing bias and precision. The mean error was 0.0477 L/h. The precision of the prediction of this parameter (0.155 L/h) remained lower than the interindividual standard deviation (0.765 L/h). This procedure enables the estimation of individual pharmacokinetic parameters for phenazone. In this study, numerous laboratory tests were performed. A highly significant correlation (p < 0.001) was found between phenazone clearance and the prothrombin time, albumin, gamma-globulin, factor V, antithrombin III, fibrinogen and total bilirubin. Discriminant analysis determined that protein, alkaline phosphatase, creatininaemia and gamma-globulin had more significant discriminating power and gave better prognostic results than those seen with the Child-Pugh test.

Glucose dehydrogenase activity in Acinetobacter species.

A study of D-glucose oxidation by Acinetobacter species was carried out. Glucose-oxidizing strains were found distributed among almost all Acinetobacter species. 14C-glucose oxidation kinetics by non-proliferating cells with separation of oxidation products (14C-gluconate) by DEAE-cellulose paper chromatography was studied. Inhibition of glucose dehydrogenase (GDH) activity by 11 carbohydrates (mono- and disaccharides) and determination of the kinetic parameters showed that glucose oxidation was due to the action of membrane-bound GDH (inactive in vivo on disaccharides). On the basis of GDH inhibition patterns obtained, two groups were individualized. The first group of strains (identified as A. calcoaceticus, A. baumannii, A. lwoffii, A. johnsonii and Acinetobacter species 3, 9, 10 and 11) showed a greater affinity for glucose than the second group (A. haemolyticus, A. junii and Acinetobacter species 6 and 12). Restoration of GDH activity after addition of pyrroloquinoline quinone (PQQ) was studied in 187 strains previously found unable to oxidize glucose. GDH activity of 150 out of 166 strains identified as A. baumannii, A. johnsonii, A. lwoffii and Acinetobacter species 11 and 12 was restored. Eighteen of 21 strains identified as A. haemolyticus and Acinetobacter species 6 were unable to produce acid from glucose after addition of PQQ. Our results confirm that the former taxonomic scheme for the genus Acinetobacter (2 species differing only by glucose oxidation) is untenable and that, accordingly, identification of Acinetobacter strains at the species level must be performed using more modern methods, i.e. carbon source utilization tests.

Transport of glucose by a phosphoenolpyruvate:mannose phosphotransferase system in Pasteurella multocida.

Pasteurella multocida was examined for glucose and mannose transport. P. multocida was shown to possess a phosphoenolpyruvate (PEP):mannose phosphotransferase system (PTS) that transports glucose as well as mannose and was functionally similar to the Escherichia coli mannose PTS. Phosphorylated proteins with molecular masses similar to those of E. coli mannose PTS proteins were visualized when incubated with 32P-PEP. The presence of an enzyme IIAGlc which could play an important role in regulation, as described in other Gram-negative bacteria, was detected. The enzymes of the pentose-phosphate pathway were present in P. multocida growth on glucose. The activity of 6-phosphofructokinase (the key enzyme of the Embden-Meyerhof pathway (EMP)), was very low in cell extracts, suggesting that EMP is not the major pathway for glucose catabolism.

Taxonomic diversity of anaerobic glycerol dissimilation in the Enterobacteriaceae.

A total of 1,123 strains representing 128 taxa in the Enterobacteriaceae (named species or subspecies and genomic species) were screened for the presence of glycerol dehydrogenases and 1,3-propanediol dehydrogenase. Only eight taxa, Citrobacter freundii sensu stricto, C. youngae, C. braakii, C. werkmanii, Citrobacter genomospecies 10 and 11, Enterobacter gergoviae and Klebsiella pneumoniae subsp. pneumoniae could grow fermentatively on glycerol and possessed both glycerol dehydrogenase type I (induced by glycerol and dihydroxyacetone) and 1,3-propanediol dehydrogenase which are typical enzymes of the anaerobic glycerol dissimilation pathway. Six other species, C. koseri, E. aerogenes, E. intermedium, K. oxytoca, K. planticola and K. terrigena could not grow fermentatively on glycerol and possessed a glycerol dehydrogenase type I but no 1,3-propanediol dehydrogenase. Other glycerol dehydrogenases types were found: type II (induced by glycerol and hydroxyacetone), type III (induced by glycerol only) and type IV (induced by hydroxyacetone only). They were widely distributed among the Enterobacteriaceae. Classification and identification may take advantage of tests exploring the dissimilation of glycerol.

Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat- or UV-killed cells.

A number of methods have been proposed to assess the viability of cells without culture. Each method is based on criteria that reflect different levels of cellular integrity or functionality. As a consequence, the interpretation of viability is often ambiguous. The purposes of this work were to evaluate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells. Methods that assess 'viability' by the demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integrity, presence of nucleic acids) or the building up of cellular material (cell elongation) have been evaluated in live and UV- or heat-killed cells. With live cells, viability markers detected cells in counts similar to the colony count. However, these so-called viability markers could stain dead cells for some time after the lethal treatment. For the UV-killed cells, residual activities were detected even after 48 h of storage at 20 degrees C. However, for heat-treated cells, these activities disappeared within hours after heat treatment. Only a combination of fluorescence in situ hybridization with rRNA probes and cell elongation in response to nutrients (in the presence of an inhibitor of cell division) had the ability to differentiate live from dead cells. Problems in the definition of a viable but nonculturable state are in part due to the lack of a clear definition of bacterial death. We consider death as an irreversible state where no growth, cell elongation or protein synthesis may occur.

Phenotypic diversity of anaerobic glycerol dissimilation shown by seven enterobacterial species.

The anaerobic glycerol pathway was studied in seven enterobacterial species selected as representative of different behaviours in terms of anaerobic glycerol dissimilation. The presence of oxidative and reductive pathways of the dha regulon in Klebsiella pneumoniae enabled the cells to grow fermentatively on glycerol. The first two enzymes of the dha regulon (glycerol dehydrogenase type I and dihydroxyacetone kinase) represent the oxidative branch, while the latter two (glycerol dehydratase and 1,3-propanediol dehydrogenase) represent the reductive branch of glycerol fermentation. The slower utilization of glycerol by K. oxytoca was attributed to low production of 1,3-propanediol. K. oxytoca lacked glycerol dehydratase and demonstrated low 1,3-propanediol dehydrogenase activity. K. planticola and K. ozaenae differed from K. pneumoniae and K. oxytoca in lacking the ability to grow on glycerol. K. planticola lacked both enzymes of the reductive branch of glycerol fermentation, and K. ozaenae possessed glycerol dehydrogenase only. K. rhinoscleromatis and Hafnia alvei, like Escherichia coli, did not possess a dha regulon. The glycerol dehydrogenase type II of H. alvei was distinct from that of E. coli. The phenotypic diversity of anaerobic glycerol dissimilation may have taxonomic applications.

Acetogenic coccoid spore-forming bacteria isolated from the rumen.

Thirteen strains of a new acetogenic bacterium were isolated from the rumen contents of lambs, llamas and bisons. This paper is the first report of Gram-positive coccoid spore-forming bacteria occurring in chains and able to use H2 + CO2 as energy source and produce acetate from this gas mixture. One of them, chosen as the reference strain for its efficiency in utilizing H2/CO2 likely via the acetyl-CoA pathway, was characterized in detail. The G + C ratio of the DNA of the organism was 46.5 mol%. The temperature and pH optimum were 37 degrees-40 degrees C and 6.3-6.8, respectively. Numerous organic substrates including some o-methylate aromatic compounds were used heterotrophically. The full 16S rRNA gene sequence was determined. The phylogeny, physiology, morphology and numerous features described here are sufficiently different from those of any bacteria described today to justify the definition of a new species. The name "New acetogenic bacterium" is temporarily proposed, awaiting a future taxonomic revision of the genus Clostridium.

Cellular activities in ultra-violet killed Escherichia coli.

In this work we analyze the physiological state of cells after lethal-UV dose disinfection using independent metabolic markers. Through the detection of some metabolic activities we proved that cell lysis does not immediately follow death in UV-irradiated Escherichia coli K12 cells.

A reproducible, simple and sensitive HPLC assay for determination of ofloxacin in plasma and lung tissue. Application in pharmacokinetic studies.

A high-performance liquid chromatographic method with fluorometric detection was developed for the analysis of ofloxacin in plasma and lung tissue. The detection was performed at 280 nm for excitation and 500 nm for emission. The procedure involves the addition of an internal standard followed by treatment of the samples with acetonitrile and dichloromethane. The proposed technique is reproducible, selective, reliable and sensitive. Linear detector response was observed for the calibration curve standards in the range of 0.1-5 micrograms ml-1 for plasma and 0.025-2.5 micrograms g-1 for lung tissue. The limit of quantitation is 5 ng ml-1 or 5 ng g-1. The accuracy of the method is good; that is, the relative error is < 10%. This method was applied to the pharmacokinetic study of ofloxacin in 24 chronic obstructive pulmonary disease patients.

[Current concept of mood disorders. Impact on the therapeutic management]. / La conception actuelle des troubles de l'humeur. Incidence sur la prise en charge thérapeutique.

The unipolar-bipolar distinction underlines the recurrence of the disorder (40 to 80% of unipolars and 95% of bipolars) and also the transition from one subtype to the other (10 to 15% of unipolars will become bipolars). Some semiological forms of depressive illness may give clues concerning the required management modalities. Depressions with psychotic features have a good response to ECT or to TCAs+neuroleptics. As other authors, Quitkin et al. find a good response of atypical depression to MAOIs. The comorbidity of mood disorders with personality disorders may be of poor prognosis. Akiskal suggested the presence of a depressive personality, Hudson and Pope suggest the notion of an affective disorders spectrum in which bulimia and OCD have a good response to serotoninergic antidepressants, whereas panic disorders have a good response to clomipramine, imipramine and MAOIs. Patient management should start with taking both the history of the disease and patient's previous treatment with a much precision as possible. Today the focus is on the particular progressive forms of resistant and chronic depressions, among which there are patients who have not received adequate treatment, and of rapid cyclers. The hypothesis of hypothyroïdism in rapid cyclers has been suggested recently. Carbamazepine and Valproate seem to be efficacious in several recent open studies and in controlled for carbamazepine. The initiation of chemotherapy to prevent the recurrences of depression takes into account the unipolar or bipolar aspect of the mood disorder. Lithium has emerged as the prophylactic agent of choice in bipolar disorders, especially if the index episode is manic. Early prophylaxis is justified when the first episode is manic or after two depressive episodes.(ABSTRACT TRUNCATED AT 250 WORDS)

[Comparison of two assessment tools of antidepressant side-effects: UKU scale versus spontaneous notification]. / Comparaison de deux outils de mesure des effets indésirables d'un traitement antidépresseur: la notification spontanée et l'échelle UKU.

BACKGROUND AND AIM OF THE STUDY: Overall, the efficacy of the newer antidepressants: serotonin selective reuptake inhibitors (SSRI), selective serotonin/norepinephrine reuptake inhibitor (SNRI), noradrenergic and specific serotonergic antidepressant (NaSSA) and tianeptine is similar to that of the tricyclics, and so their acceptability/safety becomes a selection criterion for the clinician. However, side-effect assessment comes up against several difficulties: distinguishing between somatic symptoms caused by the depression and those caused by the treatment -- which assessment tool to use (spontaneous notification, standardized scales that are not specific for the side effects caused by psychotropic drugs, standardised scales specific for the side effects caused by psychotropic drugs, meta-analysis, etc.) -- which data sources to consult (anecdotal reports, reviews, prospective studies), and which data set to use, etc. As a result, the question of the exhaustiveness and reliability of the data consulted by the clinician can arise. We therefore conducted a comparative study in patients treated with these newer antidepressants, of 2 antidepressants side-effect assessment tools: spontaneous notification (SN) versus the UKU scale, a standardised scale specific for the side effects of psychotropic drugs. METHODOLOGY: The depressed outpatients were selected from a psychiatric unit in a French psychiatric hospital and from a non-hospital consulting room. The main inclusion criteria were: male or female subjects, suffering from major depression without melancholia or psychotic features or suffering from mood disorders (according to DSM IV criteria), who had been treated for at least 4 weeks with one of the newer antidepressants. The main exclusion criteria were: any other psychiatric disorder, a serious physical disorder, treatment with neuroleptics, mood-changing drugs or other antidepressants, and patients who were not able to understand the questionnaire. The investigation was carried out by a clinical pharmacist. RESULTS: Fifty patients were included in the study. There were 18 men and 32 women. The mean age was 53.5 15.9 years [22 - 77], the mean period of treatment was 24 30.5 months [1 - 127] and 52% of the patients received concomitant medication with anxiolitic or hypnotic drug(s). The percentage of patients who reported at least one side effect was significantly higher for the UKU scale than for SN (84% vs 58%, p<0.01). The ratio between SN and UKU scale scores was 2/3. A similar pattern was found for the total number of side effects (n=177 vs n=47, p<0.001). The ratio between the total number of side effects for the SN and UKU scale was 1/4. The side effects were divided into five subgroups: psychiatric, neurovegetative, sexual, neurological and others. In all these subgroups, the number of side effects reported was significantly higher when the UKU scale was used than when SN was used. The values were as follows: psychiatric (n=44 vs n=15, p<0.001), neurovegetative (n=59 vs n=15, p<0.001), sexual (n=36 vs n=10, p<0.001), neurological (n=11 vs n=2, p<0.001) and other side effects (n=27 vs n=5, p<0.001). Nineteen side effects were only reported when SN was used (for example: dry eyes, incompatibility with alcohol, euphoria...). Twenty-four side effects were only reported when the UKU scale was used (for example: increased libido, loss of bodyweight...). The side effects had no impact on daily life in most of 80% of the patients; there was no significant difference between the patient's assessment of the discomfort caused by side effects and the clinician's assessment. In 90% of cases, the side effects did not lead to any change in the treatment. DISCUSSION: The findings of this study show that the collection of data regarding side effects depends on the assessment tool used: the number of side effects reported was significantly higher when the UKU scale was used than when SN was used. However, this finding must viewed with caution, because it has been showed that checklists can induce symptoms in suggestible patients. Neurovegetative troubles are the most commonly reported side effects, and neurological troubles the least often reported. This matches the tolerability profile of these antidepressants. The disorders that were least frequently spontaneously reported were the neurological, sexual and "other" side effects. These emerged only when the clinician asked the patient about them. The 19 side effects that were only reported when SN was used were side effects that were not included in the UKU scale or that had not been present during the three days before we started the investigation. The 34 side effects that were only reported when the UKU scale was used were either side effects with no apparent link with the treatment (for example: micturition troubles) or embarrassing effects (such as increased libido). CONCLUSION: Our findings show that the collection of data on side effects depends on the assessment tool used. These findings need to be confirmed by large-scale comparative studies, and the standardization of the assessment of side effects is a question that needs to be raised.

[Role of electroshock therapy in puerperal mental disorders]. / Place de l'électrochocthérapie dans les troubles psychiatriques de la puerpéralité.

Ten cases of psychotic patients requiring electroconvulsivotherapy (ECT) are reported during pregnancy and puerperium. The interest and the safety of ECT are confirmed. ECT is principally indicated in severe cases and in typical or atypical mood disorders.

Extracellular oxidation of D-glucose by some members of the Enterobacteriaceae.

Extracellular D-glucose oxidation by 5 enterobacterial species was studied with the purpose of selecting conditions useful for taxonomic studies. Extracellular production of gluconate from 14C-glucose by bacterial cells was evidenced by DEAE-cellulose paper chromatography. Escherichia coli oxidized glucose only when pyrroloquinoline quinone (PQQ) was added, whereas Serratia marcescens, Yersinia frederiksenii, Erwinia cypripedii and Cedecea lapagei oxidized D-glucose without added PQQ. 2-Deoxyglucose was found to be an excellent non-metabolized analogue of D-glucose in oxidation experiments. D-glucose oxidation was inhibited by KCN, p-chloromercuribenzoic acid and carbonyl cyanide m-chlorophenylhydrazone; and activated by p-benzoquinone. Iodoacetate had no action. Comparative cellulose thin-layer chromatography including 2-ketogluconate and 2,5-diketogluconate (produced by Janthinobacterium lividum) as standards, showed that gluconate was oxidized to 2-ketogluconate by S. marcescens and E. cypripedii, and 2-ketogluconate was oxidized to 2,5-diketogluconate by E. cypripedii. The diversity of D-glucose oxidation products in the Enterobacteriaceae could have some taxonomic applications.