Clinical predictors and microbiology of ventilator-associated pneumonia in the intensive care unit: a retrospective analysis in six Italian hospitals.

The purpose of this study was to assess the main clinical predictors and microbiological features of ventilator-associated pneumonia (VAP) in the Intensive Care Unit (ICU) environment. This work is a retrospective analysis over one year from September 2010 to September 2011. Patients' risk factors, causes of admission, comorbidities and respiratory specimens collected in six Italian ICUs were reviewed. Incidence and case fatality rate of VAP were evaluated. After stratification for VAP development, univariate and multivariate analyses were performed to assess the impact of patients' conditions on the onset of this infection. A total of 1,647 ICU patients (pts) were considered. Overall, 115 patients (6.9 %) experienced at least one episode of VAP. The incidence rate for VAP was 5.82/1,000 pts-days, with a case fatality rate of 44.3 %. Multivariate analysis showed that admission for neurological disorders (aIRR 4.12, CI 1.24-13.68, p = 0.02) and emergency referral to ICU from other hospitals (aIRR 2.11, CI 1.03-4.31, p = 0.04) were associated with higher risk of VAP, whereas a tendency to a higher risk of infection was detected for admission due to respiratory disease, cardiac disease, trauma and for having obesity or renal failure. A total of 372 microbiological isolates from respiratory specimens were collected in VAP patients. The most common species were Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, showing high resistance rates to carbapenems. Neurological disorders and emergency referral at the admission into the ICU are significantly associated with the onset of VAP. A high incidence of multi-drug resistant Gram- species was detected in the respiratory specimens.

The combined use of VIGI@ct (bioMérieux) and fluorescent amplified length fragment polymorphisms in the investigation of potential outbreaks.

Even with good surveillance programmes, hospital-acquired infections (HAIs) are not always recognized and this may lead to an outbreak. In order to reduce this risk, we propose a model for prompt detection of HAIs, based on the use of a real-time epidemiological information system called VIGI@ct (bioMèrieux, Las Balmas, France) and on the rapid confirmation or exclusion of the genetic relationship among pathogens using fluorescent amplified length fragment polymorphism (f-AFLP) microbial fingerprinting. We present the results of one year's experience with the system, which identified a total of 306 suspicious HAIs. Of these, 281 (92%) were 'confirmed' by clinical evidence, 16 (5%) were considered to be simple colonization and the latter nine (3%) were archived as 'not answered' because of the absence of the physician's cooperation. There were seven suspected outbreaks; of these, f-AFLP analysis confirmed the clonal relationship among the isolates in four cases: outbreak 1 (four isolates of Pseudomonas aeruginosa), outbreak 2 (three Escherichia coli isolates), outbreak 6 (two Candida parapsilosis isolates) and outbreak 7 (30 ESbetaL-producing Klebsiella pneumoniae subsp. pneumoniae). Based on our results, we conclude that the combination of VIGI@ct and f-AFLP is useful in the rapid assessment of an outbreak due to Gram-positive or Gram-negative bacteria and yeasts.

Linezolid as rescue drug: a clinical case of soft tissue infection caused by a Staphylococcus aureus strain resistant in vivo to teicoplanin.

The authors report and discuss a patient admitted to intensive care unit (ICU) for acute respiratory failure due to upper airway obstruction caused by face and neck soft tissue infection. An oxacillin-resistant Staphyloccoccus aureus was isolated from necrotic skin lesions and from skin biopsy. The strain was susceptible in vitro to teicoplanin, but it showed resistance in vivo, despite appropriate dosage. After 6 days of full dose therapy, since the clinical course worsened, teicoplanin was interrupted and linezolid was started. In 48 hours signs of infection regressed, and the patient was discharged from the ICU after 10 days of linezolid treatment. Linezolid resulted as a rescue drug for a life-threatening infection.

Aldehyde dehydrogenase from rat intestinal mucosa: purification and characterization of an isozyme with high affinity for gamma-aminobutyraldehyde.

In rat adrenal gland and gastric mucosa putrescine is efficiently oxidized to GABA via gamma-aminobutyraldehyde (ABAL) by action of diamine oxidase and aldehyde dehydrogenase. Having turned our attention on the rat intestinal mucosa, where putrescine uptake and diamine oxidase are active, we have purified and characterized an aldehyde dehydrogenase optimally active on gamma-aminobutyraldehyde. A dimer with a subunit molecular weight of 52,000, the native enzyme binds ABAL and NAD+ with high affinity: at pH 7.4, Km values are equal to 18 and 14 microM, respectively. Affinity for betaine aldehyde is much lower (Km = 285 microM), but the efficiency is equally good, thanks to a high value of V. Unaffected by disulfiram and Mg2+, the enzyme is activated by high NAD+ concentrations (Vnn = 1.6 x Vn) and is competitively inhibited by NADH. According to the best fitting model, the dimeric enzyme only binds one NADH and the mixed complex enzyme-NAD(+)-NADH is inactive. The increase of activity promoted by NAD+ can therefore be ascribed to an allosteric effect, rather than to the activation of a second reaction center. Highly stable at pH 6.8 in the presence of dithiothreitol and high phosphate concentrations, ABALDH is inactivated by ion-exchange resins and by cationic buffers. Our results show that the enzyme can be effectively involved in the metabolism of biogenic amines and, with a K(m) for ABAL lower than 20 microM, in the synthesis of GABA.

Purification and kinetic characterization of gamma-aminobutyraldehyde dehydrogenase from rat liver.

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to gamma-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD(+)-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3-8.4. Temperatures higher than 28 degrees C promote slow activation and the process is favoured by the presence of at least one substrate. Km for aliphatic aldehydes decreases from 110 microM for ABAL and acetaldehyde to 2-3 microM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD+ is affected by the aldehyde present at the active site: Km for NAD+ is approximately 70 microM with ABAL, approximately 200 microM with isobutyraldehyde and capronaldehyde, and > 800 microM with acetaldehyde. The kinetic behaviour at 37 degrees C is quite complex; according to enzymatic models, NAD+ activates the enzyme (Kact approximately 500 microM) while NADH competes for the regulatory site (Kin approximately 70 microM). In the presence of high NAD+ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (Kact approximately 10 mM). The data show that the enzyme is probably an E3 aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.

Isolation of Clostridium difficile from human jejunum: identification of a reservoir for disease?

The possibility that the small intestine may represent a reservoir for Clostridium difficile was studied, using segments of human jejunum collected at necropsy. Our results (three of 100 specimens positive for C difficile culture) support the hypothesis that C difficile can be found in human jejunum and that it adheres to the normal mucosa as a resident bacterium. These findings suggest that gastrointestinal disease caused by C difficile has an endogenous origin.

Two-year surveillance on fluconazole susceptibility of Candida spp isolates in a general and university hospital in Rome.

Fluconazole susceptibility was tested in 385 clinical yeast isolates (285 Candida albicans, 38 C. glabrata, 31 C. tropicalis, 31 other Candida subsp.) using the agar disk diffusion test. Yeasts were collected from specimens obtained from outpatients (69) and inpatients (intensive care unit: 79 isolates, major burn unit: 31 isolates, hematology ward: 45 isolates, gynecology ward: 67 isolates, other wards: 94 isolates). Three hundred and fifty-six (92%) yeast isolates showed to be susceptible, 18 (5%) were susceptible dose-dependent, and 10 (3%) were resistant to fluconazole. Of the resistant group, 3 isolates were C.albicans, while seven were Candida non-albicans (2 C. rugosa, 2 C. humicola, 1 C. tropicalis, 1 C. ciferrii, 1 C. glabrata). The disk-diffusion method was easy to perform and there were no difficulties in the interpretation of inhibition zone diameters. Fluconazole maintained a good activity against Candida spp despite its extensive use for the prophylaxis and treatment of fungal infections.

Evidence for cross-infection in an outbreak of Clostridium difficile-associated diarrhoea in a surgical unit.

Environmental studies were performed in a hospital outbreak of Clostridium difficile-associated diarrhoea. Transmission was associated with the sluice room and the storage room where medical equipment was found to be contaminated with C.difficile. Typing of isolates by antibiotic-susceptibility patterns and profiles of EDTA-extracted proteins showed the presence of an "epidemic" strain common to the majority of patients and environmental sites. Control of the outbreak was achieved by improvement of environmental hygiene and use of disposable equipment.

The activity 'in vitro' of trospectomycin against high-level antibiotic-resistant enterococci.

Trospectomycin, a new aminocyclitol antibiotic, was uniformly active against 69 isolates of enterococci with high-level resistance to steptomycin (54 isolates), gentamicin (27 isolates), ampicillin (19 isolates), ciprofloxacin (17 isolates), vancomycin (3 isolates), or teicoplanin (3 isolates). In time-killing studies, trospectomycin alone demonstrated no bactericidal activity. No synergistic interaction was demonstrated when trospectomycin was combined with ampicillin, vacomycin or ciprofloxacin.

Long-term intramuscular teicoplanin treatment of chronic osteomyelitis due to oxacillin-resistant Staphylococcus aureus in outpatients.

Oxacillin-resistant staphylococci are the most serious pathogens in chronic osteomyelitis and only glycopeptides have been shown to be efficacious against them. We assessed the safety and efficacy of a regimen of teicoplanin 400 mg/day i.m. as long-term treatment in outpatients with osteomyelitis. A total of 76 patients received teicoplanin. Twenty-five patients had chronic prosthetic osteomyelitis (20 hip) and 51 patients had osteomyelitis caused by osteo-synthesis devices. Oxacillin-resistant Staphylococcus aureus was isolated in pure culture in 55 patients (72%). A total of 21 patients had polymicrobial infection with a total of 48 isolated strains. All patients were treated with teicoplanin 400 mg i.m. once-a-day alone or with other drugs for a minimum of 4 months. Only one patient had side effects requiring discontinuation of treatment. The teicoplanin dose was reduced to 200 mg/day i.m. in 2 patients to decrease creatinine clearance values. Seventy out of 76 patients were cured.

Rat testis mitochondrial aldehyde dehydrogenase: kinetic evidence for a direct reaction between capronaldehyde and enzyme-bound NAD+ in the presence of Mg2+ ions.

In the presence of Mg2+ saturation curves of aldehyde dehydrogenase show a sharp maximum at capronaldehyde concentrations lower than 1 microM. Since the native enzyme is a dimer, kinetic data have been analyzed with a general rate equation (given as a ratio of two polynomials) that takes into account the presence of two binding sites for both substrates and two for Mg2+. Simulation of the saturation curves was only successful after allowing the formation of the stable complexes ES, ES2, ES2M, ES2M2, EM and EM2. Since ESM and ESM2 are highly reactive but very unstable, activity at low aldehyde concentration can be explained by assuming a direct reaction mediated by Mg2+. At concentrations higher than 1 microM, capronaldehyde effectively binds to the enzyme in a highly cooperative process, but the formation of ES2M and ES2M2 results in slower reaction rates. Since ES2M2 is inactive, increase of the Mg2+ concentration eventually leads to strong inhibition. Experiments at different NAD+ concentrations show that the enzyme binds two NAD+, but reaction takes place at one binding site.

An enzymatic model for rat liver alcohol dehydrogenase in the presence of low-Km aldehyde dehydrogenase.

Four isoenzymes of aldehyde dehydrogenase were partially purified from rat liver mitochondria by hydroxylapatite chromatography and gel filtration. While three forms display low affinity for acetaldehyde, the fourth is active at extremely low aldehyde concentrations (Km less than or equal to 2 microM) and allows the oxidation of the acetaldehyde formed by catalysis of alcohol dehydrogenase at pH 7.4. Different models of alcohol dehydrogenase have been examined by analysis of progress curves of ethanol oxidation obtained in the presence of low-km aldehyde dehydrogenase. According to the only acceptable model, when the acetaldehyde concentration is kept low by the action of aldehyde dehydrogenase, NADH no longer binds to alcohol dehydrogenase, but acetaldehyde still competes with ethanol for the active site of the enzyme. The seven kinetic parameters of the two enzymes (four for alcohol dehydrogenase and three for aldehyde dehydrogenase) and the equilibrium constant of the reaction catalyzed by alcohol dehydrogenase have been determined by applying a new fitting procedure here described.

Comparative study of glucosidases from the thermophilic fungus Thermoascus aurantiacus Miehe. Purification and characterization of intracellular beta-glucosidase.

Intracellular beta-glucosidase was extracted from the mycelium of Th. aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and purified to electrophoretic homogeneity. Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM. With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C. beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins. Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%).

A simple steady state kinetic model for alcohol dehydrogenase. Use of reaction progress curves for parameters estimation at pH 7.4.

A simple rate equation for alcohol dehydrogenase was obtained by assuming independent binding sites for ethanol and NAD+ and fully competitive inhibition by the products of the reaction, acetaldehyde and NADH. A random binding order was also assumed. The rate equation is described by six parameters: four association constants (two for the substrates and two for the products of the reaction), Vf for the forward direction, and the equilibrium constant of the reaction. The six parameters were determined at pH 7.4 by numerical analysis of progress curves of reactions started with different concentrations of ethanol and NAD+. The parameters for alcohol dehydrogenase partially purified from rat liver were: Km for ethanol = 0.746 mM, Km for NAD+ = 0.0563 mM, Km for acetaldehyde = 7.07 microM, Km for NADH = 4.77 microM and Keq = 2.36 X 10(-4). The computed values allowed a very good simulation of the experimental progress curves and little variation was observed in the kinetic parameters when the reactions were started in the presence of either NADH or acetaldehyde.

Kinetic properties and mechanism of action of an intracellular beta-glucosidase from Thermoascus aurantiacus Miehe.

Intracellular beta-glucosidase is strongly inhibited by its own substrate p-nitrophenyl-beta-glucoside which displays high affinity for two binding sites. A non-productive complex is formed also by cellobiose, but its lower affinity results in a much lower inhibition. As shown by inhibition experiments performed with glucono-delta-lactone, the hydrolytic reaction proceeds through the formation of a carbonium ion, very similar in its half-chair conformation to the delta-lactone. Carboxylic groups (pK = 3.19) appear involved in the catalytic process together with a histidine residue (pK = 5.64): while the carboxylate ions stabilize the carbonium ion, the displaced group accepts a proton from the protonated imidazole.

Mechanism of action and structural properties of an intracellular alpha-glucosidase from Thermoascus aurantiacus Miehe.

The intracellular alpha-glucosidase purified from the mycelium of Th. aurantiacus is an exceptionally stable protein which displays its maximum activity at 70 degrees C and pH 4.2 and is inhibited by 4 M urea, 0.5 M mercaptoethanol, 15 mM Cu++ and 0.04% rose bengal only after incubation at high temperature (60-70 degrees C). Carboxylic groups with pKa = 3.25 appear involved in the catalytic process together with a histidine residue (pKa = 5.7). Plots of Log V vs pH also show that the carboxylic groups dissociate in a cooperative way. A simple reaction mechanism is proposed on the basis of competitive inhibition by delta-gluconolactone, which suggests the formation of a carbonium ion.

Multiple forms of intracellular alpha-glucosidase from Thermoascus aurantiacus Miehe: purification and properties.

The intracellular alpha-glucosidase extracted from the mycelium of Thermoascus aurantiacus was resolved by chromatographic procedures on DEAE-cellulose and hydroxylapatite into 4 major forms which account for 96% of the recovered activity. These forms, purified to electrophoretic homogeneity, display the same affinity toward p-nitrophenyl-alpha-glucoside (Km = 0.24 mM at pH 4 and 70 degrees C), the same molecular weight on Sephadex G-200 (140,000-145,000) and isoelectric point (4.2) and are protected by Cl- and H+ ions from heat denaturation at 70 degrees C. They differ in the carbohydrate content which varies between 14 and 31%, w/w.

Catabolism of ornithine in chicken liver.

It is shown that most ornithine in a chicken liver homogenate is decarboxylated in the particulate fraction. This fraction, however, requires the cytosol for complete activity. The dialyzed supernatant does not activate decarboxylation of ornithine, while the supernatant is more effective when previously inactivated at 100 degrees C. The supernatant can be substituted by the intermediates of the citric acid cycle (oxaloacetate, citrate, succinate, malate), by pyruvate, and partially by ADP as well. Rotenone blocks decarboxylation suggesting that this occurs through the pathway ornithine leads to glutamic semialdehyde leads to glutamate leads to alpha-ketoglutarate, which in turn is decarboxylated. The activating metabolites would thus have a role in reoxidizing NADH, and the ketoacids also in supplying the acceptor for transamination of glutamate, and indirectly for ornithine transamination. Pyruvate and oxaloacetate do not transaminate with ornithine. Insulin promotes a marked increase of cytosol ornithine decarboxylase activity, but has little effect on decarboxylation by the particulate cellular fraction.

Characterization of a pyruvate dehydrogenase modulator purified from insulin-treated rat brain plasma membranes.

A factor able to stimulate pyruvate dehydrogenase when added to purified mitochondria was prepared from the supernatant of brain plasma membranes incubated with physiological concentrations of insulin (25 microU/ml). The factor completely reactivated pyruvate dehydrogenase previously inhibited with ATP and was active on pyruvate dehydrogenase from brain and liver mitochondria and from peripheral lymphocytes. The insulin-dependent stimulator of pyruvate dehydrogenase was heat and acid stable, was not absorbed on charcoal and displayed an isoelectric point of 5.5. The insulin mediator was purified by gel filtration, DEAE-cellulose and sulfonated polystyrene chromatography and, after dansylation, by high performance liquid chromatography. The purified mediator displayed a molecular weight of about 2800 and appeared as a peptide rich in glycine and serine and void of proline and sulfur containing aminoacids. It retained its stimulatory action on pyruvate dehydrogenase after dansylation and was completely inactivated by trypsin and chymotrypsin. Full reactivation of ATP-inhibited pyruvate dehydrogenase was attained when mitochondria were incubated with a mediator concentration of about 0.5 microM.

[Antibiotic sensitivity of bacterial strains isolated in the course of lower urinary tract infections. Comparison between two case series in different years]. / Sensibilità chemio-antibiotica di ceppi batterici isolati in corso di infezioni delle basse vie urinarie. Confronto tra 2 casistiche in anni differenti.

Antibiotic susceptibility on 803 strains isolated from urine in 1990 and on 500 strains isolated in 1978 have been tested. The comparison between the results on strains of the two periods confirm the utility of urine culture for cost effective treatments.