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PubMed2003

Frequency of isolation and antimicrobial susceptibility of bacterial pathogens isolated from patients with bloodstream infections: a French prospective national survey.

All bloodstream strains, total 1463, isolated during a 1 month period in 105 hospitals representing all geographical areas in France were collected to study their antimicrobial susceptibility. The three major species were Escherichia coli, Staphylococcus aureus and coagulase-negative staphylococci. Among the 242 S. aureus, 87 were resistant to methicillin and among those 99% were resistant to ciprofloxacin, 11.5% to gentamicin, 1% to quinupristin/dalfopristin and 8% were heterogeneously resistant to vancomycin. Study of the methicillin-resistant S. aureus indicated that 12 clones had disseminated in French hospitals, six being heterogeneously resistant to vancomycin. Among the Streptococcus pneumoniae, 43% showed decreased susceptibility to the penicillins and 42% to erythromycin. One isolate was highly resistant to fluoroquinolones. Gentamicin, cefotaxime, ciprofloxacin and gatifloxacin resistance was rare in Enterobacteriaceae with 95% of strains susceptible. The incidence of extended-spectrum beta-lactamases was quite low. Moreover more than 25% of Pseudomonas aeruginosa strains were resistant to ciprofloxacin and gentamicin. The magnitude of antibiotic resistance in bloodstream isolates, in particular Gram-positive bacteria, emphasizes the importance of hospital control measures, rational prescribing policies and new vaccine strategies.

Anti-Infective Agents↗
PubMed1995

Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ.

By extensively examining the experimental conditions for time-resolved spectrophotometry of non-transparent light scattering systems, we demonstrated the feasibility of quantitative analysis of both the fluorescence lifetime and intensity of reduced pyridine nucleotides in living tissues, suspensions of isolated liver mitochondria, and hepatocytes, as well as hemoglobin-free perfused rat liver being used systematically for measurements. The fluorescence decay was analyzed by the maximum likelihood method with a 4-component decay model. The lifetime of NADH observed in mitochondria (mean: 2.8 +/- 0.2 ns) was much longer than that of the free form in an aqueous solution (mean: 0.43 +/- 0.01 ns), and it was characterized as a protein-bound form. The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy state, though the intensity changed markedly. The decay curves of isolated hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superimposed. Under the conditions of "unphysiological" acidosis, the mean lifetime became about 1.5 times longer than that under normal conditions. With perfused liver, the relative contributions of cytosolic NADH and NADPH were determined by infusing lactate and tert-butylhydroperoxide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides. In contrast, about 70% of the total fluorescence intensity was due to cytosolic NADPH, but its decay parameters were essentially the same as those of mitochondrial NADH. No free form of either NADH or NADPH was detected in the cytosolic and mitochondrial spaces. We concluded that the changes in fluorescence intensity observed under the various conditions can be simply explained by a change in the amount of reduced pyridine nucleotides in tissues, rather than by changes in the microscopic environment. The wide applicability of time-resolved fluorescence photometry to in vivo studies is well documented.

Aerobiosis↗
PubMed2002

Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation, as assessed by the Edmonton Isolation Protocol.

BACKGROUND: Current techniques for isolating islets require that pancreata stored with University of Wisconsin solution (UW) are processed within 12 hours of cold storage. In this study, we hypothesized that the two-layer method (TLM) could extend the acceptable preservation period of pancreata before islet isolation and increase islet yields. METHODS: In the first experimental set, eight pancreata were maintained for an average of 8.3+/-1.2 hours in UW and transferred into the TLM for an additional 14.3+/-1.1 hours for a total cold ischemic period of 22.6+/-1.6 hours (prolonged TLM). Four pancreata were maintained as a control group in UW alone for a total of 21.3+/-2.0 hours. In the second experimental set, six pancreata were maintained for an average of 6.4+/-1.8 hours in UW followed by 4.8+/-0.8 hours with the TLM for a total preservation time of 11.3+/-2.5 hours (short TLM). The control organs for the short TLM group were stored for an average of 9.5+/-1.3 hours in UW alone. Islets were isolated and evaluated according to the Edmonton protocol. RESULTS: Between each group of the two experimental sets, there was no significant difference in donor-related factors (i.e. gender, age, body mass index [BMI], etc.). The TLM as compared with UW preservation resulted in a significant increase in islet yields postpurification for both short (3,353+/-394 islet equivalents [IE] vs. 2,027+/-415 IE; mean+/-SEM) and prolonged (2,404+/-503 IE vs. 514+/-180 IE) periods of storage. Furthermore, islet yields after prolonged storage with the TLM were not significantly different from organs maintained for only a short period with UW (P=0.17). The quality of islets as assessed by size, postculture viability, survival rates, insulin content, and insulin secretion were similar for each of the four groups. CONCLUSION: In comparison with UW organ preservation, exposure of pancreata to the TLM result in greater islet yields and extended preservation times.

Adenosine↗
PubMed1997

Melphalan dosing regimens for management of recurrent melanoma by isolated limb perfusion: application of a physiological pharmacokinetic model based on melphalan distribution in the isolated perfused rat hindlimb.

The optimal dosing schedule for melphalan therapy of recurrent malignant melanoma in isolated limb perfusions has been examined using a physiological pharmacokinetic model with data from isolated rat hindlimb perfusions (IRHP). The study included a comparison of melphalan distribution in IRHP under hyperthermia and normothermia conditions. Rat hindlimbs were perfused with Krebs-Hen-seleit buffer containing 4.7% bovine serum albumin at 37 or 41.5 degrees C at a flow rate of 4 ml/min. Concentrations of melphalan in perfusate and tissues were determined by high performance liquid chromatography with fluorescence detection. The concentration of melphalan in perfusate and tissues was linearly related to the input concentration. The rate and amount of melphalan uptake into the different tissues was higher at 41.5 degrees C than at 37 degrees C. A physiological pharmacokinetic model was validated from the tissue and perfusate time course of melphalan after melphalan perfusion. Application of the model involved the amount of melphalan exposure in the muscle, skin and fat in a recirculation system was related to the method of melphalan administration: single bolus > divided bolus > infusion. The peak concentration of melphalan in the perfusate was also related to the method of administration in the same order. Infusing the total dose of melphalan over 20 min during a 60 min perfusion optimized the exposure of tissues to melphalan whilst minimizing the peak perfusate concentration of melphalan. It is suggested that this method of melphalan administration may be preferable to other methods in terms of optimizing the efficacy of melphalan whilst minimizing the limb toxicity associated with its use in isolated limb perfusion.

Animals↗
PubMed1992

Complete nucleotide sequence of RNA 4 of rice stripe virus isolate T, and comparison with another isolate and with maize stripe virus.

The complete nucleotide sequence of RNA 4 of rice stripe virus isolate T (RSV-T) was determined and found to consist of 2157 nucleotides, containing two open reading frames (ORFs). One, deduced to be present in the 5'-proximal region of the viral-sense RNA, encodes the stripe disease-specific protein with Mr 20541, and the other ORF, in the 5'-proximal region of the viral complementary sense RNA, encodes an unknown protein with Mr 32474. Between these two ORFs there is an intergenic non-coding region that could form a secondary structure with two base-paired hairpin configurations. These characteristics indicate that RSV-T RNA 4 has an ambisense coding strategy. Comparison of the two ORFs of RSV-T with those of another isolate revealed 97.2% and 98.0% identity for the nucleotide sequences, and 98.3% and 98.2% identity for the amino acid sequences. The leader sequences of these two isolates were the same. However, an insertion was found in the intergenic non-coding region of RSV-T. Furthermore, comparison of the nucleotide and amino acid sequences of RSV-T RNA 4 with those of RNA 4 of maize stripe virus, which is another member of the tenuivirus group, revealed greater identity, suggesting a close phylogenetic relationship between these two viruses.

Amino Acid Sequence↗
PubMed2000

Tripartite haemolysin BL: isolation and characterization of two distinct homologous sets of components from a single Bacillus cereus isolate.

Haemolysin BL (HBL), a three-component enterotoxic/necrotizing/vascular permeability toxin, is a likely virulence factor of Bacillus cereus diarrhoeal food poisoning and necrotic infections. This paper describes the isolation of two distinct homologous sets of all three HBL components from a single B. cereus isolate, MGBC 145. The proteins of one set (designated HBL, consisting of B, L(1) and L(2)), were about 87-100% identical in N-terminal amino acid sequences to their respective prototype components from strain F837/76, and the proteins of the homologous set (HBL(a), consisting of B(a), L(1a) and L(2a)) were all about 62-65% identical. Only the latter homologues differed immunochemically and physicochemically from the prototypes. HBL and HBL(a) exhibited similar haemolytic and vascular permeability potencies, and the homologues could be interchanged freely. There were no notable differences in activity between the L component homologues. However, components B and B(a) were significantly different. Both were secreted as monomers, but unlike B, B(a) was isolated as a relatively inactive complex that could be reactivated with urea. When B(a) was substituted for B in gel-diffusion assays the distinct discontinuous haemolysis pattern typical of the presence of B did not occur. In suspension assays, excess B inhibited the haemolysis of B-primed cells by L(1) (as previously described), but not that of B(a)-primed cells. Excess B(a) had the opposite effect and enhanced lysis of B(a)-primed cells, but not that of B-primed cells. These differences reveal details about how the toxin components interact on target cell membranes. The authors' observations indicate that HBL represents a new family of multicomponent toxins that was generated by a process of gene and operon duplication that occurred either intracellularly or by horizontal transfer, and raise the possibility of the existence of other related toxins in the genetically diverse B. cereus taxonomic group.

Amino Acid Sequence↗
PubMed1980

Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY.

Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [(3)H]UTP into a trichloroacetic acid-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [(3)H]yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 millimolar (NH(4))(2)SO(4), 100 millimolar KCl), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [(3)H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro.

Journal Article↗
PubMed1984

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products.

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans, five were B. circulans and four were B. licheniformis. Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65 degrees C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70 degrees C for 30 min.

Animals↗
PubMed1984

Glutathione conjugation by isolated lung cells and the isolated, perfused lung. Effect of extracellular glutathione.

Cells isolated from rat lung by protease digestion were found to catalyze the reduced glutathione (GSH) conjugation of 1-chloro-2,4-dinitrobenzene. The rate of conjugation was stimulated severalfold in the presence of GSH, indicating uptake and utilization of extracellular GSH by the lung cells. The stimulation was dependent on the GSH concentration and not due to a spontaneous nonenzymatic reaction or to extracellular GSH-transferase activity. Conjugation of 1-chloro-2,4-dinitrobenzene was also measured using isolated perfused rat lung. The conjugation, which was linear for a longer time than with the isolated cells, was also stimulated in the presence of GSH in the perfusion medium. The results indicate the ability of rat lung to utilize extracellular GSH.

Animals↗
PubMed1996

Pharmacological studies on prostanoid receptors in the rabbit isolated saphenous vein: a comparison with the rabbit isolated ear artery.

1. In isolated ring preparations of the rabbit saphenous vein, prostaglandin E2 (PGE2) caused well-defined, stable and concentration-dependent relaxations of KCl contracted tissues with a mean potency (p[A50]) of 9.39. 2. The prostanoid EP-receptor agonists, PGE1, 11-deoxy PGE1, 16,16-dimethyl PGE2 and misoprostol were all full agonists in this preparation. The EP2-receptor selective agonists, butaprost and AH13205, and the EP1/EP3-receptor selective agonist, sulprostone, also relaxed this tissue but were at least 300 times less potent than PGE2. 3. Prostaglandin D2 (PGD2), the DP-receptor agonist, BW245C, and the IP-receptor agonist, cicaprost, caused concentration-dependent relaxations of the rabbit saphenous vein but were at least 60 times less potent than PGE2. 4. The selective EP4-receptor antagonist, AH23848B (30 microM), antagonized the PGE2 concentration-effect (E/[A]) curves yielding a pA2 estimate of 4.96. The EP1/DP-receptor antagonist, AH6809 (10 microM), had no effect on the location of PGE2 E/[A] curves. 5. The stable thromboxane A2-mimetic, U46619, elicited concentration-dependent contractions of the rabbit saphenous vein (p[A50] = 8.01) however, PGE2 and prostaglandin F2 alpha (PGF2 alpha) were unable to produce a contractile response. The response to U46619 was competitively antagonized by the TP-receptor antagonist, GR32191B, yielding a pKB estimate of 7.08. 6. In the rabbit isolated ear artery, PGE2, misoprostol and AH13205 relaxed tissues pre-contracted with phenylephrine. PGE2 (p[A50] = 7.04) and misoprostol were equipotent, whereas AH13205 was some 40 fold less potent. AH23848B (30 microM) and AH6809 (1 and 10 microM) caused no significant shift in the location of PGE2 E/[A] curves. 7. These data suggest that the rabbit isolated saphenous vein contains prostanoid, EP-, DP-, IP- and TP-receptors. Based on antagonist affinity information and agonist potency orders, the rabbit saphenous vein contains an inhibitory prostanoid EP-receptor different from that in the rabbit ear artery, but comparable to the recently described EP4-receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

Comparison of pig hepatocyte isolation using intraoperative perfusion without warm ischemia and isolation of cells from abattoir organs after warm ischemia.

Enzymatic hepatocyte isolation using warm ischemic pig livers from an abattoir was compared with isolation using in situ perfused organs. Using organs from animals of 30 kg body mass (BM), the intraoperative perfusion showed superior results. The use of livers from abattoir pigs of 40-50 kg BM after warm ischemia resulted in a lower yield of hepatocytes and in high rates of injured cells. The mean yield in the intraoperative perfusion group was 68 +/- 11% (wet weight), the maximum yield in the abattoir organ group was 58%. The mean viability in the intraoperative perfusion group was 65 +/- 14% (trypan blue) compared with a maximum viability of 39% in the abattoir liver group. Additional purification by density gradient centrifugation improved the viability of the abattoir liver group to a mean of 95% (trypan blue). The use of pig livers from large abattoir animals required additional purification steps to improve viability since the cell yield is considerably lower than with intraoperative organ perfusion. In general, hepatocyte isolation from abattoir organs is not recommended.

Abattoirs↗
PubMed2006

Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol.

BACKGROUND: Circulating endothelial cells (CECs) have been identified as markers of vascular damage in a variety of disorders, such as myocardial infarction, vasculitis, and transplantation. CD146-driven immunomagnetic isolation has gained widespread use, but the technique is hampered by the lack of a definition of CECs and the absence of a consensus for their enumeration. AIM: To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration. METHODS: We devised a protocol based on CD146-driven immunomagnetic isolation and a subsequent confirmatory step with Ulex-Europaeus-Lectin-1 staining. In a multi-center effort, we evaluated the preanalytical and analytical phases of this protocol. We evaluated the effects of storage, anticoagulation and density centrifugation, and compiled previous experience with this technique. RESULTS: Our protocol permitted unequivocal identification of CECs with acceptable reproducibility. There was an effect of storage time in that median cell numbers declined to only 87.5% of their baseline values during 24 h of storage at 4 degrees C. Recovery was lower with citrate than with ethylene-diamine tetra-acetic acid after 4 h of storage; density centrifugation was also associated with lower recovery. We provide a comprehensive list of technical recommendations and potential pitfalls. Finally, based on our experience with this protocol and a recent consensus workshop, we formulated a working definition for CECs. CONCLUSION: Our work represents an important step toward consensus regarding the CECs. Our recommendations represent the experience of three major centers and should now be scrutinized by others in the field.

CD146 Antigen↗
PubMed1999

Characterization of the antiseptic-resistance gene qacE delta 1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1.

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.

Amino Acid Sequence↗
PubMed1992

[Isolation of vero-cytotoxin-producing Escherichia coli from cattle and serotyping and toxin-typing of the isolated strains].

Two hundred and sixty-six piglets with diarrhea (from 4 farms), 73 healthy pregnant pig (from 2 farms), 27 calves with diarrhea (from 9 farms) and 47 healthy milk cows (from 1 farm) were examined for Vero-cytotoxin-producing Escherichia coli (VTEC), and 52, 11, 15 and 67 strains of VTEC were isolated from 17 piglets, 11 pregnant pigs, 6 calves and 23 milk cows, respectively. All VTEC strains from the piglets produced only VT2vp, while the strains from the healthy pigs did not produce VT2vp, but did VT1 and/or VT2. Most VTEC strains from calves and cows produced VT2vhb and some produced VT2 and VT1. Serotyping of the isolated strains showed that many strains from the piglets belonged either O139:H1, O141:H4 or O141:HUT, but the strains from the pigs were either R-form or O-untypable. Many strains from the calves and cows were serotyped into O116 or O113, but there were several R-form and O-untypable. From these results, it is suggested that VTEC strains, especially from the pregnant pigs, calves with diarrhea and healthy milk cows, which produced the same type of Verotoxins to that produced by human isolates, may become sources of human infections.

Animals↗
PubMed1997

[Isolation frequency and biological characteristics of the multiple-antibiotic resistant Pseudomonas aeruginosa isolated from clinical specimens].

Isolation frequency of multiple-antibiotic resistant Pseudomonas aeruginosa (MARPA) was 11.9% (fifty six strains) of a total of four-hundred seventy-one strains of P. aeruginosa isolated from clinical specimens at the Kyorin University Hospital from October 1994 to December 1996. Eighteen strains of MARPA and thirteen strains of antibiotic sensitive P. aeruginosa (ASPA) isolated from clinical specimens in internal medicine ward A were determined O serotype, and characterized with production of pyocyanin, pyoverdin, hemolysin, elastase, and caseinase. Sixteen strains (88.9%) of MARPA were identified as serotype C. The ability to produce pyocyanin, hemolysin, elastase, and caseinase was not detected in all MARPA. One side, thirteen strains of ASPA showed various serotypes, i.e., B: 5 strains (38.4%), G: 4 strains (30.8%), C: 2 strains (15.4%), E: 1 strain (7.7%) and unknown type: 1 strain (7.7%), and the production of both hemolysin and pyoverdin was observed in 13 strains (100%), pyocyanin in 8 strains (61.5%), elastase and caseinase in 9 strains (69.2%) of ASPAs, which suggests that ASPAs do maintain the synthetic ability of pathogenic factors and pigments, but MARPAs do not. These results indicate that from epidemiological points of view the current strains of MARPA spread from one clone within the internal medicine ward A with nosocomial outbreak by serotype C.

Ampicillin Resistance↗
PubMed2000

[Isolation of Aeromonas species from patients with sporadic diarrhea and characterization of Aeromonas hydrophila isolates].

A total of 16 strains of Aeromonas species were isolated from feces of 348 patients with sporadic diarrhea in western Kanagawa, Japan from 1996 to 1998. Of the 16 isolates, 7 were Aeromonas hydrophila, 1 was A. sobria and 8 were A. caviae. The strains of A. hydrophila were examined for hemolytic activities, hemolysin gene types and O-serogroups. Although all 7 strains of A. hydrophila showed hemolytic activities on sheep blood agar, in the test for hemolytic activities in culture supernatant, only 1 of the these strains showed no hemolytic activity against sheep erythrocytes. From the results of PCR assay, the tested strains of A. hydrophila were grouped into 2 hemolysin gene types of [ahh1 + ahh3 + aerA] (n = 6) and [ahh1 + aerA] (n = 1) both of which are recognized to be enteropathogenic. Five of the 7 strains of A. hydrophila belonged to serogroup O11. These results suggest that 7 strains of A. hydrophila isolates are recognized to be enteropathogenic strains and serogroup O11 is the major O-serogroup of enteropathogenic A. hydrophila in humans.

Aeromonas↗
PubMed1990

Comparative in vitro activity of cefixime against Haemophilus influenzae isolates, including ampicillin-resistant, non-beta-lactamase-producing isolates, from pediatric patients.

The in vitro activity of cefixime was comparatively tested against 232 non-type b and 102 type b isolates of Haemophilus influenzae derived from clinical specimens of pediatric patients, including 10 non-type b strains that did not produce beta-lactamase and demonstrated resistance to ampicillin. Cefixime was active against the ampicillin-susceptible and ampicillin-resistant, beta-lactamase-producing isolates; however, its activity against some non-beta-lactamase-producing, ampicillin-resistant isolates appeared to be limited.

Ampicillin Resistance↗
PubMed1996

Synergistic inhibition of human immunodeficiency virus isolates (including 3'-azido-3'-deoxythymidine-resistant isolates) by foscarnet in combination with 2',3'-dideoxyinosine or 2',3'-dideoxycytidine.

The combinations of foscarnet plus 2',3'-dideoxyinosine and foscarnet plus 2',3'-dideoxycytidine synergistically inhibit the replication of human immunodeficiency virus type 1 isolates, including two 3'-azido-3'-deoxythymidine-resistant isolates. The combination of 2',3'-dideoxyinosine plus 2',3'-dideoxycytidine showed additive inhibition of the majority of the human immunodeficiency virus isolates tested. All three combinations showed pronounced antagonistic cytotoxicity and thus were less toxic to the growth of peripheral blood mononuclear cells than the separate drugs.

Antiviral Agents↗