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.

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.

Agmatine induces apoptosis in rat hepatocyte cultures.

BACKGROUND/AIMS: Agmatine, the compound formed by decarboxylation of arginine, is believed to be an endogenous neurotransmitter through interaction with the imidazoline receptors. However, it also appears to regulate rat hepatocyte polyamines by modifying both their synthesis and their catabolism. As the decrease in polyamine content has been correlated with apoptosis, we examined the possibility that agmatine has an effect on this phenomenon. METHODS: Apoptotic cells were detected by visualizing nuclear shrinkage/fragmentation in hepatocytes cultured at 21 and 5% oxygen tension. Caspase-3 activity, cleavage of PARP, release of cytochrome c and mitochondrial swelling were therefore measured in the two conditions and in the presence or not of agmatine. RESULTS: In rat hepatocytes agmatine promoted apoptosis, procaspase 3 processing and increase of caspase-3 like activity. This occurred through mitochondria swelling and release of cytochrome c. Cyclosporin A and catalase blocked the swelling. CONCLUSIONS: Our experiments show that agmatine, besides all the known biological effects, has also part, at least in hepatocytes, in the modulation of programmed cell death.

[Interstitial pulmonary fibrosis in chronic HCV hepatitis treated with interferon]. / Fibrosi polmonare interstiziale in corso di epatite cronica da HCV in trattamento con interferone.

The study describes the clinical, virologic and immunological characteristics of a patient with interstitial pulmonary fibrosis during interferon treatment for HCV chronic hepatitis. After the interruption of the interferon and the beginning of immunosuppressive treatment, an improvement of pulmonary pathology was observed. At the reintroduction of interferon, the patient presented a rapid worsening of pulmonary fibrosis with a normalization of biochemical and virologic parameters of hepatitis. The correlation among interstitial pulmonary fibrosis, HCV infection and interferon treatment is discussed; however in the described case, the pulmonary pathology was correlated to interferon treatment.

Transport and metabolism of agmatine in rat hepatocyte cultures.

Rat hepatocytes in culture take up [14C]-agmatine by both a high-affinity transport system [KM = 0.03 mM; Vmax = 30 pmol x min x (mg protein)-1] and a low-affinity system. The high-affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4-guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C-4-aminobutyrate is also accumulated in the presence of an inhibitor of 4-aminobutyrate transaminase.

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.

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.

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.

Casein kinase 2 phosphorylates recombinant human spermidine/spermine N1-acetyltransferase on both serine and threonine residues.

Casein kinase 2 purified from human erythrocyte cytosol has been found to phosphorylate human spermidine/spermine N1-acetyltransferase (SSAT) expressed as a fusion protein in E. coli and purified to homogeneity with a specific activity similar to that reported for pure human SSAT. The amino acid sequence of the protein revealed not less than four phosphorylable residues, optimal target for protein kinase 2 phosphorylation being flanked by acid residues in position +1 and +3. Our results indicate that most 32P-phosphate is taken up by Ser residues, as evidenced by HCl hydrolysis and electrophoresis and that the phosphorylation extent is modulated by the physiological polyamine concentration. Partial digestion with trypsin at a low concentration for less than one hour preferentially hydrolyzes Lys-Arg-Arg in position 141-143 of the SSAT suggesting that the Ser-phosphorylated residues are located in the C-terminus of the protein, probably Ser 146 and 149.

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.

Differentiation of Clostridium difficile, Clostridium bifermentans, Clostridium sordellii, and Clostridium perfringens from diarrheal stool by API ZYM and API LRA oxidase test.

A simple, rapid and reliable outline for identification of clostridia isolates from human infections was developed. It consists of a combination of API ZYM and API LRA Oxidase tests. The enzymatic activities were performed with strains sub-cultured onto carbohydrate-free medium (Columbia blood agar). Fifty-five strains of Clostridium difficile, C. bifermentans, C. sordellii, and C. perfringens from clinical specimens and eight reference standard strains representing different species of the same genus were analyzed. The accuracy of the new method was evaluated by comparison with the results obtained by DNA/DNA analysis.

[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.

Regulation by progesterone and pregnenolone of dimeric aldehyde dehydrogenase from rat testis cytoplasm.

1. Aldehyde dehydrogenase from rat testis cytosol has been purified to electrophoretic homogeneity. With an isoelectric point of 9.5, the enzyme appears a dimer with a subunit molecular weight of 52,500. 2. The influence of pregnenolone and progesterone on the kinetic behaviour has been investigated using valeraldehyde as substrate. 3. The kinetic data were fitted to a modified version of the Monod-Wyman-Changeux model and the fitting procedure resulted in a good correspondence between theoretical and experimental reaction rates over a wide range of valeraldehyde concentrations. 4. According to the model, the dimeric enzyme is in equilibrium between two conformational states R and T. The R state displays higher affinity for valeraldehyde, but lower catalytic power. In the absence of substrates and effectors the [T]/[R] ratio is near to 1. 5. Pregnenolone and progesterone activate the enzyme by stabilizing the more active state T and by increasing the catalytic power of the R state. The increase of activity is counteracted by the inhibition exerted by both steroids on the T state.

In vitro activity of a new antibacterial drug, trospectomycin sulphate (U-63,366F), against Bacteroides strains isolated from the vagina.

The antibacterial activity of trospectomycin, clindamycin, metronidazole, imipenem, cefoxitin, and piperacillin was tested against 72 Bacteroides spp. strains isolated from the vagina of women with vaginitis by determining the minimal inhibitory concentration using the agar dilution method. Trospectomycin shows a good activity which is comparable to that of imipenem and metronidazole. Its expanded spectrum of activity makes trospectomycin suitable for the use in single-drug therapy of pelvic infections in women.

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.

Allosteric regulation of rat testis mitochondrial aldehyde dehydrogenase by capronaldehyde and magnesium ion.

1. The influence of Mg2+ on the kinetic behaviour of mitochondrial aldehyde dehydrogenase from rat testis has been investigated using capronaldehyde as substrate. 2. The kinetic data, obtained by numerical analysis of the progress curves of aldehyde oxidation, were fitted to a modified version of the Monod-Wyman-Changeux model and the fitting procedure resulted in a good correspondence between theoretical and experimental reaction rates over a wide range of capronaldehyde and Mg2+ concentrations. 3. According to the model, the tetrameric enzyme is in equilibrium between two conformational states R and T which display comparable affinities for capronaldehyde (the dissociation constants are 0.17 and 0.3 microM, respectively), but different catalytic power (VT = 2VR). The T state can bind with lower affinity a second molecule of aldehyde (K = 2.5 microM). 4. Mg2+ stabilizes the T state (the dissociation constants for the R and T states are 2.2 and 0.12 mM, respectively) and acts as a strong activator of the R state, but as a weak inhibitor of the T state. In the absence of substrates and Mg2+, the R<-->T equilibrium favors the R state ([T]/[R] = 0.16). 5. The model is able to predict the kinetic behaviour also when the NAD+ concentrations are not saturating and when inhibitory effects by NADH are taken into account.

Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).