Am J Respir Cell Mol Biol, 2001 Dec, 25(6), 699 - 706 Effects of surfactant protein A and NaCl concentration on the uptake of Pseudomonas aeruginosa by THP-1 cells; Khubchandani KR et al.; In the present study, we characterized a model system in which we examined the effects of human surfactant protein A (SP-A) on the uptake of a common human pulmonary pathogen, Pseudomonas aeruginosa, by a human monocytic/macrophage cell line, THP-1 cells . We found that SP-A significantly increases uptake of the bacteria in a dose-dependent manner . Bacterial uptake was temperature-dependent, because an effect of SP-A on bacterial uptake was observed at 37 degrees C and not at 4 degrees C . The continued presence of SP-A during the period when the bacteria and THP-1 cells were co-incubated was necessary for enhanced uptake . Pre-incubation of the bacteria or THP-1 cells with SP-A, followed by washing, abolished the effect of SP-A on bacterial uptake . The effect of SP-A could be inhibited by high concentrations of mannose, but was not affected by the removal or addition of lipopolysaccharide (LPS) . Finally, we observed that the SP-A-mediated increase in uptake of P . aeruginosa by THP-1 cells was optimal in a narrow (100 mM and 150 mM) range of NaCl concentrations . We conclude that SP-A enhances the THP-1 cell-mediated uptake of P . aeruginosa in a manner dependent on temperature, the concentration of SP-A, and the concentration of NaCl. Proc Natl Acad Sci U S A, 2001 Dec 4, 98(25), 14613 - 8 Epub 2001 Nov 27. A quorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism; Cao H et al.; The human opportunistic pathogen Pseudomonas aeruginosa strain PA14 infects both plants and animals . Previously, using plants to screen directly for P . aeruginosa virulence-attenuated mutants, we identified a locus, pho34B12, relevant in mammalian pathogenesis . Here, nonsense point mutations in the two opposing ORFs identified in the pho34B12 locus revealed that one of them, mvfR (multiple virulence factor Regulator), is able to control all of the phenotypes that mutant phoA34B12 displays . Both genetic and biochemical evidence demonstrate that the mvfR gene encodes a LysR-like transcriptional factor that positively regulates the production of elastase, phospholipase, and of the autoinducers, 3oxo-dodecanoyl homoserine lactone (PAI I) and 2-heptyl-3-hydroxy-4-quinolone (PQS), as well as the expression of the phnAB operon, involved in phenazine biosynthesis . We demonstrate that the MvfR protein is membrane-associated and acts as a transcriptional activator until cells reach stationary phase, when a unique negative feedback mechanism is activated to signal the down-regulation of the MvfR protein . This work reveals an unprecedented virulence mechanism of P . aeruginosa and identifies a unique indispensable player in the P . aeruginosa quorum-sensing cascade. Appl Environ Microbiol, 2001 Dec, 67(12), 5648 - 55 Integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry and molecular cloning for the identification and functional characterization of mobile ortho-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2; Hickey WJ et al.; Protein mass spectrometry and molecular cloning techniques were used to identify and characterize mobile o-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2 and Pseudomonas huttiensis strain D1 . Proteins induced in strains JB2 and D1 by growth on 2-chlorobenzoate (2-CBa) were extracted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry . Two bands gave significant matches to OhbB and OhbA, which have been reported to be the alpha and beta subunits, respectively, of an ortho-1,2-halobenzoate dioxygenase of P . aeruginosa strain 142 (T . V . Tsoi, E . G . Plotnikova, J . R . Cole, W . F . Guerin, M . Bagdasarian, and J . M . Tiedje, Appl . Environ . Microbiol . 65:2151-2162, 1999) . PCR and Southern hybridization experiments confirmed that ohbAB were present in strain JB2 and were transferred from strain JB2 to strain D1 . While the sequences of ohbA from strains JB2, D1, and 142 were identical, the sequences of ohbB from strains JB2 and D1 were identical to each other but differed slightly from that of strain 142 . PCR analyses and Southern hybridization analyses indicated that ohbAB were conserved in strains JB2 and D1 and in strain 142 but that the regions adjoining these genes were divergent . Expression of ohbAB in Escherichia coli resulted in conversion of o-chlorobenzoates to the corresponding (chloro)catechols with the following apparent affinity: 2-CBa approximately 2,5-dichlorobenzoate > 2,3,5-trichlorobenzoate > 2,4-dichlorobenzoate . The activity of OhbAB(JB2) appeared to differ from that reported for OhbAB(142) primarily in that a chlorine in the para position posed a greater impediment to catalysis with the former . Hybridization analysis of spontaneous 2-CBa(-) mutants of strains JB2 and D1 verified that ohbAB were lost along with the genes, suggesting that all of the genes may be contained in the same mobile element . Strains JB2 and 142 originated from California and Russia, respectively . Thus, ohbAB and/or the mobile element on which they are carried may have a global distribution. Bull Exp Biol Med, 2001 Apr, 131(4), 356 - 60 Electron microscopic study of DNA packing in phi(kz) bacteriophage (Pseudomonas Aeruginosa) irradiated with laser in the presence of a dye; Manykin AA et al.; DNA packing of bacteriophage phikz (Pseudomonas aeruginosa) was studied by electron microscopy after gentle destruction of the capsid with powerful laser impulse in aqueous virus suspension in the presence organic dye . Suspension of destroyed phage heads contained complexes of genomic DNA with endogenous capsid proteins characterized by pronounced regular structure . The type of these structures corresponds to one the main model of phage DNA packing, the so-called coil packing. Environ Sci Technol, 2001 Nov 1, 35(21), 4242 - 51 Transition from cometabolic to growth-linked biodegradation of vinyl chloride by a Pseudomonas sp . isolated on ethene; Verce MF et al.; Pseudomonas aeruginosa strain DL1 was isolated on ethene as a sole carbon and energy source . When ethene-grown DL1 was first exposed to vinyl chloride (VC), the rate of VC consumption was very rapid and then declined sharply, indicative of a cometabolic process . A lack of growth and significant release of soluble products during this interval also indicates that the initial activity on VC was cometabolic . Following the rapid initial rate of VC cometabolism, a slow rate of VC utilization continued . After an extended period of incubation (>40 days), a transition occurred that allowed DL1 to begin using VC as a primary growth substrate, with an observed yield, maximum growth rate, and Monod half saturation coefficient of 0.21 mg of total suspended solids/mg VC, 0.046 d(-1), and 1.17 microM VC, respectively, at 22 degrees C . Acetylene inhibits consumption of ethene and VC by ethene-grown cells, suggesting a monooxygenase is responsible for initiating metabolism of these alkenes . Resting cells grown on ethene cometabolized VC with an observed transformation capacity of 9.1 micromol VC/mg total suspended solids and a transformation yield of 0.22 mol VC/mol ethene . The presence of 40 microM ethene increased the rate and amount of VC cometabolized . However, consumption of higher concentrations of ethene decreased the total amount of VC consumed, and VC inhibited ethene utilization . A kinetic model was developed that describes substrate interactions during batch depletion of ethene and VC for a range of initial concentrations . The results suggest that ethene may stimulate in situ biodegradation of VC either by functioning as a primary substrate to support cometabolism of VC or by selecting for organisms that can utilize VC as a primary substrate. Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13972 - 7 Molecular basis for defective glycosylation and Pseudomonas pathogenesis in cystic fibrosis lung; Poschet JF et al.; The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF) . The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known . Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells . The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P . aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases . Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN . These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments. J Bacteriol, 2001 Dec, 183(24), 7126 - 34 Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice; Hendrickson EL et al.; We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14 . A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r) . PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source . Compared to wild-type PA14, rpoN::Gen(r) was ca . 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans . In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P . aeruginosa PA14 at later stages of infection . rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model . These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P . aeruginosa pathogenicity in diverse hosts. J Am Chem Soc, 2001 Nov 28, 123(47), 11623 - 31 Electron tunneling in single crystals of Pseudomonas aeruginosa azurins; Crane BR et al.; Rates of reduction of Os(III), Ru(III), and Re(I) by Cu(I) in His83-modified Pseudomonas aeruginosa azurins (M-Cu distance approximately 17 A) have been measured in single crystals, where protein conformation and surface solvation are precisely defined by high-resolution X-ray structure determinations: 1.7(8) x 10(6) s(-1) (298 K), 1.8(8) x 10(6) s(-1) (140 K), {Ru(bpy)2(im)(3+)-}; 3.0(15) x 10(6) s(-1) (298 K), {Ru(tpy)(bpy)(3+)-}; 3.0(15) x 10(6) s(-1) (298 K), {Ru(tpy)(phen)(3+)-}; 9.0(50) x 10(2) s(-1) (298 K), {Os(bpy)2(im)(3+)-}; 4.4(20) x 10(6) s(-1) (298 K), {Re(CO)3(phen)(+)} (bpy = 2,2'-bipyridine; im = imidazole; tpy = 2,2':6',2' '-terpyridine; phen = 1,10-phenanthroline) . The time constants for electron tunneling in crystals are roughly the same as those measured in solution, indicating very similar protein structures in the two states . High-resolution structures of the oxidized (1.5 A) and reduced (1.4 A) states of Ru(II)(tpy)(phen)(His83)Az establish that very small changes in copper coordination accompany reduction but reveal a shorter axial interaction between copper and the Gly45 peptide carbonyl oxygen {2.6 A for Cu(II)} than had been recognized previously . Although Ru(bpy)2(im)(His83)Az is less solvated in the crystal, the reorganization energy for Cu(I) --> Ru(III) electron transfer falls in the range (0.6-0.8 eV) determined experimentally for the reaction in solution . Our work suggests that outer-sphere protein reorganization is the dominant activation component required for electron tunneling. Arch Biochem Biophys, 2001 Dec 1, 396(1), 111 - 8 Kinetic mechanism and pH dependence of the kinetic parameters of Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase; Naught LE et al.; The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) is responsible for the formation of mannose 1-phosphate and glucose 1-phosphate in the human pathogenic bacterium Pseudomonas aeruginosa . Mannose 1-phosphate and glucose 1-phosphate are required for the biosynthesis of polysaccharides that contribute to the virulence of P . aeruginosa, so inhibitors of PMM/PGM may lead to clinically useful compounds . The V/K values for mannose 6-phosphate and glucose 6-phosphate show that they are equally good substrates for the enzyme . PMM/PGM overexpressed in Escherichia coli is isolated as a phosphoenzyme; surprisingly, mutation of serine 108 where phosphorylation occurs results in phosphorylation of a different residue so that activity is reduced only 20-fold from that of wild-type enzyme . In the reverse reaction glucose 1-phosphate exhibits substrate inhibition, which arises through its competition with the activator glucose 1,6-bisphosphate for binding to dephosphoenzyme . This phenomenon is consistent with a mechanism in which the enzyme phosphorylates the substrate to generate a bisphosphorylated intermediate that reorients in the active site to return its original phosphoryl group to the enzyme and generate the observed product . The pH dependence of the kinetic parameters suggests that the active site contains a residue that serves as a general base in the catalytic reaction and one that acts as a general acid . However, the pK of the general acid is 7.4 and that of the general base is 8.4 so these residues exist in a state of reverse protonation in the active enzyme . (c)2001 Elsevier Science. Zhonghua Yi Xue Za Zhi, 1999 Dec, 79(12), 908 - 10 {Treatment of invasive burn wound infection with sepsis: a clinical study}; Chai J et al.; OBJECTIVE: To further improve the treatment of burn wound sepsis . METHODS: Eight patients with burn wound sepsis, of whom 6 with MODS and two with septic shock, were treated consecutively in our hospital from September 1997 to October 1998 . The plasma concentrations of IL-6, IL-8 TNF and LPS were assayed before and after surgical intervention and at the time when the patients' vital signs became stable . RESULTS: (1) The patients' conditions abruptly deteriorated when the burn wound sepsis emerged . (2) The major causative factor related to burn wound sepsis was extensive burn injuries, with large area of deep burn remained open . (3) Although colonization by multiple pathogenic bacteria was found, Pseudomonas aeruginosa was the most frequent bacteria isolated from the subeschar tissue . (4) The plasma concentrations of IL-6, IL-8, TNF and LPS before surgical intervention were significantly higher than those after surgical intervention (P < 0.05); (5) The lowest level of the inflammatory mediators were observed when the conditions of patients became stable, and the values were significantly lower compared with those before surgical intervention (P < 0.001) . CONCLUSIONS: The main cause of burn wound sepsis is the presence of a large area of infected open deep burn wounds, which should be excised and covered early . LPS and pro-inflammatory mediators play an important role in the pathogenesis of burn wound sepsis . Favorable results in the treatment attribute to appropriate application of multiple treatments, and early, aggressive and thorough surgical excision of invasive burn infectious tissue and closure of wounds play a crucial role. Pharmacotherapy, 2001 Nov, 21(11), 1320 - 4 Susceptibility of pseudomonas aeruginosa to cefepime versus ceftazidime in patients with cystic fibrosis; Robinson CA et al.; STUDY OBJECTIVES: To compare the susceptibility of respiratory cultures of Pseudomonas aeruginosa obtained from patients with cystic fibrosis to cefepime versus ceftazidime . The pattern of cumulative resistance of P aeruginosa to cefepime in patients who had received at least one treatment course of cefepime between two sputum cultures was also characterized . DESIGN: Prospective consecutive data collection . SETTING: University-affiliated cystic fibrosis clinic and medical center . PATIENTS: Eighty patients with cystic fibrosis who had at least one sputum culture positive for P aeruginosa with reported microbiologic susceptibilities to cefepime and ceftazidime . INTERVENTION: Patient data was collected and analyzed . Measurements and Main Results . Two hundred and thirty-one P aeruginosa isolates were collected over 6 months . A total of 16.4% and 8.7% of the isolates were nonsusceptible to cefepime and ceftazidime, respectively (p=0.01) . In eight patients who had not received cefepime before the study period, nonsusceptibility was 11.8% and 27.2% before and after exposure to cefepime, respectively . CONCLUSIONS: Susceptibility of P . aeruginosa isolates in patients with cystic fibrosis was lower with cefepime than with ceftazidime . Follow-up surveillance to determine changes in susceptibility of P aeruginosa isolates to cefepime is warranted. Eur J Clin Microbiol Infect Dis, 2001 Sep, 20(9), 657 - 60 Comparison of antimicrobial use and resistance of bacterial isolates in a haematology ward and an intensive care unit; Lang A et al.; The objective of the study presented here was to compare antimicrobial use and resistance of bacterial isolates in the haematology ward and the intensive care unit of Bolzano General Hospital . The bacterial organisms isolated most frequently from patients in the two wards (coagulase-negative staphylococci, Enterococcus spp., and Pseudomonas aeruginosa) were investigated for antimicrobial resistance . Isolates obtained from patients in the haematology ward were more often resistant to antimicrobial agents than isolates obtained from patients in the intensive care unit, and the agents against which the highest rates of resistance were found were third- and fourth-generation cephalosporins, carbapenems and monobactams, quinolones, aminoglycosides, and trimethoprim-sulfamethoxazole . These classes of antimicrobial agents were also used more frequently in the haematology ward than in the intensive care unit . Conversely, penicillinic beta-lactam antibiotics, rifamycins, macrolides and lincosamides were used less frequently in the haematology ward than in the intensive care unit, and the rates of resistance against these classes of antimicrobial agents were significantly lower in the haematology ward than in the intensive care unit . The results support the hypothesis that a causal relationship exists between antimicrobial use and the development of resistance and indicate that careful monitoring of antimicrobial use in hospitals is required to identify situations in which prescription patterns are contributing to the development of resistance. Afr J Med Med Sci, 2000 Sep-Dec, 29(3-4), 281 - 3 Antimicrobial activity of Mallotus oppositifolium extractives; Ogundipe OO et al.; A bioactivity monitored phytochemical examination of the morphological parts of Mallotus oppositifolium utilizing the hole-in-plate bioassay procedure against gram-positive, gram-negative bacteria and fungal isolates resulted in the location of significant antimicrobial activity in the acidic fraction (HAF) of the hexane extract of the powdered leaves . Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were 32.5 microg/ml and 65 microg/ml against Pseudomonas aeruginosa NCTC 6750 and 25 microg/ml and 50 microg/ml against Staphylococcus aureus NCTC 6571 respectively . These activities were found comparable with standard drugs. ORL J Otorhinolaryngol Relat Spec, 2001 Nov-Dec, 63(6), 333 - 40 Hearing loss in relation to round window membrane morphology in experimental chronic otitis media; Nordang L et al.; The present study was performed to test the effect of single and repeated Pseudomonas aeruginosa exotoxin A (PaExoA) instillations in the middle ear of the rat . The hearing level was examined by the ABR technique, round window membrane (RWM) thickness was measured and morphology was studied by light microscopy . The results showed both reversible and permanent hearing loss (HL) . In animals that received a single dose of PaExoA, the RWM thickness doubled initially and remained thickened during the observation period . When PaExoA was instilled on several occasions, RWM thickness doubled, before decreasing to near-control levels . This study confirms the toxicity of PaExoA and the partially reversible HL occurring after a single application of the toxin . The diminished effect of repeated toxin instillations--despite the decreasing thickness of the RWM--is discussed . Antimicrob Agents Chemother, 2001 Dec, 45(12), 3468 - 73 Population pharmacokinetics and use of Monte Carlo simulation to evaluate currently recommended dosing regimens of ciprofloxacin in adult patients with cystic fibrosis; Montgomery MJ et al.; Pharmacodynamic data on ciprofloxacin indicate that a target area under the concentration-time curve from 0 to 24 h (AUC(0-24))/MIC ratio of >or=125 is necessary to achieve optimal bactericidal activity for the treatment of gram-negative pneumonia . The purpose of this prospective study was to (i) develop a pharmacokinetic (PK) model to be utilized for therapeutic drug monitoring (TDM) of ciprofloxacin and (ii) evaluate current ciprofloxacin dosing regimens for pneumonias in cystic fibrosis (CF) patients . Twelve adult CF patients received a single 400-mg dose of IV ciprofloxacin . Six blood samples were obtained over a 12-h interval . Serum drug concentrations were determined by high-pressure liquid chromotography and were fitted to one- and two-compartment models by using NPEM2 . Ciprofloxacin MIC data for Pseudomonas aeruginosa were obtained from 1,213 CF patients enrolled in a large clinical trial . A Monte Carlo simulation was performed to estimate the fractional attainment of an AUC(0-24)/MIC ratio of >or=125 . A two-compartment model best describes the serum drug concentration data . The mean fitted PK parameter values are volume of distribution in the central compartment, 0.29 liter/kg; volume of distribution at steady state, 1.1 liters/kg; total clearance, 0.34 liter/h/kg; distributional clearance, 0.89 liter/h/kg; half-life at alpha phase, 0.16 h; and half-life at beta phase, 2.9 h . The overall fractional attainment of achieving an AUC(0-24)/MIC ratio of >or=125 against P . aeruginosa isolates with ciprofloxacin (400 mg every 12 h {q12h} and 8 qh) were 10 and 30%, respectively . A clinical breakpoint MIC of <0.5 microg/ml for susceptibility is suggested, based on an examination of the fractional attainment of the AUC(0-24)/MIC target at each MIC . The recommended doses of 400 mg q8h or q12h may be inadequate to treat an acute pulmonary exacerbation when given alone . The poor and variable AUC(0-24)/MIC ratios support the use of TDM to monitor and adjust the dosage to optimize the efficacy of ciprofloxacin therapy in these patients. Enferm Infecc Microbiol Clin, 2001 Nov, 19(9), 432 - 4 {The activity of four fluoroquinolones against strains of Pseudomonas aeruginosa with a different sensitivity pattern to ceftazidime and imipenem}; Pascual A A et al.; OBJECTIVES: To evaluate the activity of four fluorquinolones (ciprofloxacin, clinafloxacin, norfloxacin and perfloxacin) against clinical strains of Pseudomonas aeruginosa with different sensitivity patterns to ceftazidime and imipenem . MATERIAL AND METHODS: 156 strains of isolated P . aeruginosa were studied at the Virgin Macarena University Hospital in Seville during 1998 and 1999 . The in vitro activity of four fluorquinolones was determined by microdilution in Mueller Hinton bouillon, supplemented with cations, following the NCCLS guidelines . RESULTS: For all the strains evaluated, the minimum inhibitory concentration values (MIC90) of the clinafloxacin (4 mg/l) were significantly less than those for ciprofloxacin (64 mg/l) . In the 76 strains resistant to ciprofloxacin, the clinafloxacin and ciprofloxacin MCI90 were 16 and >128 mg/l respectively . Clinafloxacin was more active than ciprofloxacin, norfloxacin and pefloxacin, independent to the sensitivity pattern or the resistance to ceptazidime and imipenem . CONCLUSION: Clinafloxacin was more active in vitro than ciprofloxacin against P . aeruginosa. Curr Opin Pulm Med, 2001 Nov, 7(6), 434 - 40 The clinical use of colistin in patients with cystic fibrosis; Beringer P; Colistin is a cationic polypeptide antibiotic from the polymyxin family that was first introduced in 1962 but abandoned in the early 1970s because of initial reports of severe toxicities . However, a recent increase in the prevalence of multidrug resistant (MDR) Pseudomonas aeruginosa and the lack of novel agents in development calls for a need to re-examine the role of colistin therapy in patients with cystic fibrosis . Current data supports the use of intravenous colistimethate for the treatment of acute pulmonary exacerbations involving MDR P . aeruginosa and inhaled therapy for initial colonization . The frequency of nephrotoxicity and severity of neurotoxicity seem to be substantially less than previously believed . In addition, recent pharmacokinetic and pharmacodynamic data suggests new intravenous dosing regimens may enhance efficacy while minimizing toxicities; such regimens deserve further evaluation . Pre- and post-treatment spirometry is recommended at initiation of inhaled colistin therapy to identify sensitized individuals . Judicious use of colistin where the benefits have been clearly documented will retain this as a useful agent in the management of P . aeruginosa infections in patients with cystic fibrosis. J Biol Chem, 2002 Feb 15, 277(7), 5322 - 9 Epub 2001 Nov 12. Protein phosphatase 2A and protein kinase Calpha are physically associated and are involved in Pseudomonas aeruginosa-induced interleukin 6 production by mast cells; Boudreau RT et al.; Pulmonary infection with Pseudomonas aeruginosa is characterized by massive airway inflammation, which comprises significant cytokine production . Although mast cells are abundant in the lung and are potent sources of various cytokines, a role of mast cells in P . aeruginosa infection remains undefined, and P . aeruginosa-induced signaling mechanisms in mast cells have not been studied previously . Here we demonstrate that human cord blood-derived mast cells, mouse bone marrow-derived mast cells, and the mouse mast cell line MC/9 produce significant amounts of interleukin 6 (IL-6) in response to P . aeruginosa . This response was accompanied by a stimulation of protein kinase Calpha (PKCalpha) phosphorylation and PKC activity and was significantly blocked by the PKC inhibitors Ro 31-8220 and PKCalpha pseudosubstrate . Interestingly, mast cells treated with P . aeruginosa had reduced protein levels of phosphatase 2A catalytic unit (PP2Ac), which prompted us to determine whether a direct association between PKCalpha and PP2A occurs in mast cells . In mouse bone marrow-derived mast cells and MC/9 cells, as well as in the human mast cell line HMC-1, PP2A coimmunoprecipitated with PKCalpha either using PKCalpha- or PP2Ac-specific antibodies, suggesting that PKCalpha and PP2Ac are physically associated in mast cells . The PP2A inhibitor okadaic acid induced P . aeruginosa-like responses in mast cells including increased PKCalpha phosphorylation, stimulated PKC activity, and augmented IL-6 production, the last being blocked by the PKC inhibitor Ro 31-8220 . Finally, okadaic acid potentiated the P . aeruginosa-induced IL-6 production . Collectively, these data provide, to our knowledge, the first evidence of both a direct physical association of PP2A and PKCalpha in mammalian cells and their coinvolvement in regulating mast cell activation in response to P . aeruginosa. Infect Immun, 2001 Dec, 69(12), 7396 - 401 Antioxidant enzyme expression in clinical isolates of Pseudomonas aeruginosa: identification of an atypical form of manganese superoxide dismutase; Britigan BE et al.; Expression of superoxide dismutases (FeSOD and MnSOD) and catalases by laboratory strains of Pseudomonas aeruginosa is modulated by exogenous factors . Whether clinical isolates behave similarly and whether antioxidant enzyme expression influences P . aeruginosa virulence remain unclear . Fifty-seven P . aeruginosa blood culture isolates, plus seven pairs of blood and local-site isolates, were examined for FeSOD, MnSOD, and catalase production in vitro . Under iron-replete growth conditions FeSOD and catalase activities were maximized . MnSOD was not detected . FeSOD and catalase activity decreased under iron-limited growth conditions, whereas MnSOD activity appeared . SOD and catalase activity did not change with site of isolation or by patient . MnSOD could not be expressed by one isolate due to a missense mutation in sodA that produced a premature stop codon . Eleven percent of the isolates expressed a novel, rapidly migrating MnSOD that was associated with missense mutations in the normal stop codon of sodA . We conclude that clinical P . aeruginosa isolates vary little in FeSOD and catalase expression . Some strains produce a newly described MnSOD variant, whereas one is deficient in MnSOD production . The absence of MnSOD expression in a P . aeruginosa strain causing invasive human disease indicates that MnSOD is probably not essential for P . aeruginosa virulence. Clin Exp Immunol, 2001 Nov, 126(2), 266 - 73 Dexamethasone impairs pulmonary defence against Pseudomonas aeruginosa through suppressing iNOS gene expression and peroxynitrite production in mice; Satoh S et al.; To elucidate the in vivo mechanisms involved in the impairment in pulmonary defence as the result of treatment with glucocorticoids, we established fatal pneumonia with bacteraemia in dexamethasone (DEX)-treated mice by means of an intratracheal challenge of Pseudomonas aeruginosa . An increased neutrophil influx was observed in bronchoalveolar lavage (BAL) fluids from both untreated and DEX-treated mice . The complete suppression of an inducible isoform of nitric oxide synthase (iNOS) mRNA expression and tumour necrosis factor alpha (TNF-alpha) production during the early phase of pneumonia, but not CXC chemokine production, were found in the case of the DEX-treated mice . An immunohistochemical study with a specific antibody also revealed negative staining for nitrotyrosine in the lung tissue of DEX-treated mice, while the formation of nitrotyrosine, which indirectly indicates the generation of peroxynitrite with a potent bactericidal activity, was detected clearly in the bronchial epithelium as well as alveolar phagocytic cells of lung tissue from untreated mice . Furthermore, an intraperitoneal administration of S-methyl-isothiourea (SMT), a potent inhibitor of NOS, significantly decreased the survival and increased bacterial density in the case of untreated mice . In contrast, no significant effects on the survival and bacterial density in the lung and blood were found as the result of treatment with SMT in DEX-treated mice . Collectively, a complete repression of iNOS gene expression and a lack of the generation of peroxynitrite as well as an inhibition of TNF-alpha production in the lung appeared to be responsible for the progression of the fatal pneumonia due to P . aeruginosa in DEX-treated mice. Arch Microbiol, 2001 Nov, 176(5), 339 - 46 Flagellin inhibits Myoviridae phage phiCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site; Geiben-Lynn R et al.; PhiCTX is a double-stranded DNA phage of the Myoviridae family that converts Pseudomonas aeruginosa into a cytotoxin producer . A 42-kDa phiCTX-inhibiting protein was purified from the outer membrane fraction of P . aeruginosa strain GuA18 by octyl-beta-glucoside extraction, DEAE-chromatography, and mono-Q HPLC . This protein had an isoelectric point of 5.4 and bound specifically {125I}-labeled phiCTX . The N-terminal amino acid sequence of six out of seven Lys-C fragments was highly similar (87%) to that of the entire of type-a flagellin of P . aeruginosa strain PAK . At a concentration of 14 nM, purified flagellin protein caused a 50% decrease in the phage titer after a 20-min incubation at 37 degrees C (PhI50) . The presence of ethanol was necessary to reconstitute the inhibitory activity . In contrast, no ethanol treatment was necessary for the inhibitory activity of the sheared flagellin filaments from P . aeruginosa strain GuA18, which consists of the 42-kDa flagellin subunits and the synthesized 17-mer phage-binding-peptide NGSNSDSERTALNGEAK, representing flagellin residues 100-116 of P . aeruginosa strain PAK . The PhI50 was 10 nM and 200 nM, respectively . Antisera against the flagellin filament protein as well as against the 17-mer peptide neutralized phage infection . These results indicated that the amino acid region 100-116 of the flagellin subunit of strain GuA18 is involved in phiCTX binding . This region might play a role in phage attachment. Biomaterials, 2001 Dec, 22(24), 3217 - 24 Bacterial adhesion to surface hydrophilic and hydrophobic contact lenses; Bruinsma GM et al.; The aim of this paper was to determine the adhesion of two physico-chemically characterized bacterial strains to a surface hydrophilic (CL A, water contact angle 57 degrees) and hydrophobic (CL B, water contact angle 106 degrees) hydrogel contact lens (CL) with and without an adsorbed tear film in a parallel plate flow chamber . Hydrophobicity (by water contact angles), charge (by particulate microelectrophoresis) and elemental composition (by XPS) of the surfaces of seven bacterial strains were characterized, after which two strains were selected for further studies . On CL surfaces, hydrophobicity, elemental composition, and mean surface roughness (by AFM) were determined, as well as the protein composition of tear films adsorbed on these lenses (by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE)) . Bacterial cell surfaces were relatively uncharged and water contact angles on lawns of different strains ranged from hydrophobic to hydrophilic . After adsorption of tear film components, N/C elemental surface concentrations increased on CL A and CL B and differences in water contact angles between both lenses reduced to range from 57 degrees (CL A) to 69 degrees (CL B) . However, different protein compositions were inferred . The surface roughness of CL A increased from 4 to 13 nm . while it remained 16 nm for CL B . Adhesion of hydrophobic Pseudomonas aeruginosa #3 was more extensive than of hydrophilic Staphylococcus aureus 799, with no differences between both lenses . The hydrophobicity of P . aeruginosa #3 after cell surface damage decreased and its adhesion was reduced on CL A and strongly on CL B . In addition, passage of an air-liquid interface yielded more detachment of S . aureus 799 than of P . aeruginosa #3 from the CL surfaces . In conclusion, the hydrophobicity of CL surfaces dictates the composition of the adsorbed tear film and therewith plays an important role in bacterial adhesion to lenses . Adhesion of hydrophobic P . aeruginosa #3 was more tenacious than of hydrophilic S . aureus 799. Biochem Biophys Res Commun, 2001 Nov 16, 288(5), 1223 - 30 Two c-type cytochromes, NirM and NirC, encoded in the nir gene cluster of Pseudomonas aeruginosa act as electron donors for nitrite reductase; Hasegawa N et al.; Three c-type cytochromes, NirM, NirC, and NirN, are encoded in the nirSMCFDLGHJEN gene cluster for cytochrome cd(1)-type nitrite reductase (NIR) of Pseudomonas aeruginosa . nirS is the structural gene for NIR . NirM (cytochrome c(551)) is reported to be a physiological electron donor for nitrite reductase . The respective functions of NirC and NirN have remained unclear . In this study, we produced recombinant NirC and NirN in P . aeruginosa, and purified them from the periplasmic fraction . N-terminal amino acid sequences of the purified proteins showed that the N-terminal 31 and 18 residues of NirC and NirN precursors were cleaved, respectively, indicating that cleaved peptides act as signals for membrane translocation . In addition, the ability of NirC for electron donation to nitrite reductase was investigated . NirC, as well as NirM, was able to mediate the electron donation from the membrane electron pathway to NIR, suggesting that the structural gene for NIR is followed by the genes for two electron donors for NIR . Curr Opin Mol Ther, 2001 Oct, 3(5), 497 - 502 Recombinant adeno-associated virus vectors for cystic fibrosis gene therapy; Flotte TR; Cystic fibrosis (CF) is an autosomal recessive inherited disorder that affects approximately 30,000 North Americans . Defects in the CF transmembrane conductance regulator (CFTR) gene lead to altered secretions from exocrine glands and the pulmonary airways, to a heightened susceptibility to airway infections with Pseudomonas aeruginosa, and to severe airway inflammation . Early attempts to develop a genetic therapy for CF have not met with great clinical success, but these efforts have driven the development of viral gene transfer technology for in vivo gene delivery . The recombinant adeno-associated virus (rAAV) system has proven to be safe for in vivo gene delivery in the airways of experimental animals and CF patients, although potential barriers to delivery have been identified . These barriers may limit the transduction efficiency of this vector, especially in the context of the inflamed airways of adolescent and adult CF patients . We anticipate that the use of alternative rAAV serotype capsids and other vector alterations, along with targeting the lungs of CF patients in the earlier stages of their disease, might eventually allow for these potential limitations to be overcome. J Immunol, 2001 Nov 15, 167(10), 5913 - 20 IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice; Faggioni R et al.; IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor . Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18 . IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay . In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml) . In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality . IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg . Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS . Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression . Given at the moment of priming with P . acnes, IL-18BP-Fc decreases P . acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS . IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab . In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18. J Immunol, 2001 Nov 15, 167(10), 5880 - 6 Therapeutic administration of anti-PcrV F(ab')(2) in sepsis associated with Pseudomonas aeruginosa; Shime N et al.; The effects of rabbit-derived polyclonal Ab against PcrV, a protein involved in the translocation of type III secreted toxins of Pseudomonas aeruginosa, was investigated in two animal models of P . aeruginosa sepsis . In a mouse survival study, the i.v . administration of anti-PcrV IgG after the airspace instillation of a lethal dose of P . aeruginosa resulted in the complete survival of the animals . In a rabbit model of septic shock associated with Pseudomonas-induced lung injury, animals treated with anti-PcrV IgG intratracheally or i.v . had significant decreases in lung injury, bacteremia, and plasma TNF-alpha and significant improvement in the hemodynamic parameters associated with shock compared with animals treated in a similar manner with nonspecific control IgG . The administration of anti-PcrV F(ab')(2) showed protective effects comparable to those of whole anti-PcrV IgG . These results document that the therapeutic administration of anti-PcrV IgG blocks the type III secretion system-mediated virulence of P . aeruginosa and prevents septic shock and death, and that these protective effects are largely Fc independent . We conclude that Ab therapy neutralizing the type III secretion system has significant potential against lethal P . aeruginosa infections. Arch Biochem Biophys, 2001 Nov 15, 395(2), 169 - 76 Structural consequences of divalent metal binding by the adenylyl cyclase toxin of Bordetella pertussis; Rhodes CR et al.; Adenylyl cyclase toxin of Bordetella pertussis has been shown by several investigators to require Ca(2+) for its actions on target cells, but little is known about the nature and specificity of divalent metal binding to this novel toxin . Calcium is the preferred divalent metal since toxic actions are markedly reduced in the presence of divalent species other than calcium . Mn(2+) EPR was used to quantitate and characterize divalent metal binding and revealed that the toxin contains approximately 40 divalent metal sites, consisting of at least one class of high-affinity sites that bind Mn(2+) with a K(D) of 0.05 to 0.35 microM and one or more classes of lower affinity sites . Water proton relaxation data indicate that approximately 30 of these sites are completely inaccessible to bulk solvent . Our observations, together with the sequence homology between adenylyl cyclase toxin and the alkaline protease of Pseudomonas aeruginosa, indicate that the formation of five beta-sheet helices within the repeat domain of the toxin upon binding Ca(2+) is required for cell intoxication . Biochemistry, 2001 Nov 13, 40(45), 13728 - 33 Copper binding before polypeptide folding speeds up formation of active (holo) Pseudomonas aeruginosa azurin; Pozdnyakova I et al.; Cofactors often stabilize the native state of the proteins; however, their effects on folding dynamics remain poorly understood . To uncover the role of one cofactor, we have examined the folding kinetics of Pseudomonas aeruginosa azurin, a small blue-copper protein with a copper cofactor uniquely coordinated to five protein residues . Copper removal produces apo-azurin which adopts a folded structure identical to that of the holo-form . The folding and unfolding kinetics for apo-azurin follow two-state behavior . The extrapolated folding time in water, tau approximately 7 ms, is in good agreement with the topology-based prediction . Copper uptake by folded apo-azurin, to govern active (holo) protein, is slow (tau approximately 14 min, 50:1 copper-to-protein ratio) . In contrast, the formation of active (holo) azurin is much faster when copper is allowed to interact with the unfolded polypeptide . Refolding in the presence of 10:1, 50:1, and 100:1 copper:protein ratios yields identical time-trajectories: active azurin forms in two kinetic phases with folding times, extrapolated to water, of tau = 10 +/- 2 ms (major phase) and tau = 190 +/- 30 ms (minor phase), respectively . Correlating copper-binding studies, with a small peptide derived from the metal-binding region of azurin, support that initial cofactor binding is fast (tau approximately 3.7 ms) and thus not rate-limiting . Taken together, introducing copper prior to protein folding does not speed up the polypeptide-folding rate; nevertheless, it results in much faster (> 4000-fold) formation of active (i.e., holo) azurin . Living systems depend on efficient formation of functional biomolecules; attachment of cofactors prior to polypeptide folding appears to be one method to achieve this. Can Respir J, 2001 Sep-Oct, 8(5), 361 - 5 Bullae, bronchiectasis and nutritional emphysema in severe anorexia nervosa; Cook VJ et al.; STUDY OBJECTIVES: Pulmonary complications of anorexia nervosa are rarely documented . The case of a patient with anorexia nervosa and pulmonary disease is presented, a new quantitative computed tomography (CT) method for the detection of emphysema is employed, the literature is reviewed and the concept of 'nutritional' emphysema is discussed . RESULTS: The case of a 34-year-old, nonsmoking woman with long-standing severe anorexia nervosa who was evaluated for cough and progressive shortness of breath is reported . Pulmonary function testing showed a predominant restrictive pattern with a marked reduction in carbon monoxide transfer and respiratory muscle strength, and an elevated residual volume . Imaging revealed bullae and bronchiectasis, and quantitative analysis of the CT scan was consistent with mild, generalized emphysema . Bronchial washings grew Pseudomonas aeruginosa . Known causes for bronchiectasis were excluded . A literature review disclosed few reported noninfectious pulmonary complications of anorexia nervosa . CONCLUSIONS: To the authors' knowledge, this is the first report of bullae and bronchiectasis in a patient with anorexia nervosa, and the CT analysis was consistent with mild emphysema . Malnutrition has been associated with emphysematous changes in animals and may be the primary insult in the development of emphysema, bullae and bronchiectasis in the present patient. Pathol Biol (Paris), 2001 Oct, 49(8), 620 - 3 {Are the prolonged carrier and interhospital clonal diffusion of serotype O12 multiresistant Pseudomonas aeruginosa connected?}; Reseau epidemiologique des utilisateurs du systeme SIR; In a recent study, patients infected or colonised with serotype O12 Pseudomonas aeruginosa remained carriers of this serotype for significantly longer periods of time than patients infected or colonised with other serotypes of P . aeruginosa . The present study in 12 centres of the REUSSIR network, confirms this ecological observation on 10,506 serotypable isolates in 7,183 patients . This finding supports other arguments published in the biomedical literature, suggesting that infected or colonised patients might be the primary reservoirs of a prevalent European clone of P . aeruginosa serotype O12 that resists to multiple antibiotics. Clin Infect Dis, 2001 Dec 1, 33(11), 1859 - 64 Epub 2001 Oct 24. Influence of previous exposure to antibiotic therapy on the susceptibility pattern of Pseudomonas aeruginosa bacteremic isolates; El Amari EB et al.; Many patients who present with Pseudomonas aeruginosa bacteremia have been previously exposed to antibiotics . To assess whether resistance of bacteremic strains to antipseudomonal antibiotics (piperacillin, ceftazidime, imipenem, ciprofloxacin, or aminoglycosides) is associated with previous exposure to these drugs, a case-control study including 267 cases of P . aeruginosa bacteremia was conducted . Twenty-five percent of the episodes had been preceded by the exposure to an antipseudomonal antibiotic . Eighty-one strains were resistant to at least 1 antibiotic; 186 were susceptible to all drugs . Via univariate analysis, the risks of resistance to ceftazidime and imipenem were found to be significantly associated with previous receipt of these agents . Using multivariate analysis, exposure to any antipseudomonal antibiotic as a monotherapy was found to be associated with an increased risk of subsequent resistance to itself (odds ratio, 2.5; P=.006) . Therefore, clinicians should avoid readministering previously prescribed antibiotics when initiating empiric therapies for possible P . aeruginosa bacteremia, especially when they have been given as monotherapies. Cochrane Database Syst Rev . 2001;(4):CD002767. Elective versus symptomatic intravenous antibiotic therapy for cystic fibrosis; Breen L et al.; BACKGROUND: Pseudomonas aeruginosa is the commonest micro-organism associated with respiratory infections in cystic fibrosis . Retrospective studies have suggested that survival is increased by using an aggressive policy of intravenous antipseudomonal antibiotics at regular intervals, irrespective of symptoms . OBJECTIVES: To determine whether there is evidence that an elective (regular) versus symptomatic intravenous antibiotic regime is associated with an improvement in clinical status and survival rates in patients with cystic fibrosis . To identify any adverse effects associated with the use of elective intravenous antibiotics, including an increase in the development of resistant organisms . SEARCH STRATEGY: The Cochrane Cystic Fibrosis and Genetics Disorders Group Specialist Trials Register was used . This comprises references identified from comprehensive electronic database searches, hand searching relevant journals and abstracts from conference proceedings . Date of the most recent search of the Group's specialised register: October 2000 . SELECTION CRITERIA: All randomised or pseudo-randomised controlled trials describing the use of elective compared with symptomatic intravenous antibiotic policies for any duration or dose regimen . Elective versus symptomatic intravenous antibiotic regimes against any organisms were considered . Patients with cystic fibrosis were of any age or disease severity . DATA COLLECTION AND ANALYSIS: Trials were independently assessed for inclusion criteria, methodological quality and data extraction by the two reviewers . MAIN RESULTS: Three trials were identified by the initial search . Two trials reporting results from a total of 79 patients were included in the review . Differences in study design and objectives meant that data could not be pooled for meta-analysis . Neither trial demonstrated significant differences in outcome measures between intervention and comparison groups . REVIEWER'S CONCLUSIONS: Studies are insufficient to identify conclusive evidence favouring a policy of elective intravenous antibiotic administration, despite its widespread use . Neither are the potential risks adequately evaluated . The results should be viewed with caution as patient numbers are small . Clearly there is a need for a well-designed, adequately powered, multi-centred randomised controlled trial to evaluate these issues. Cochrane Database Syst Rev . 2001;(3):CD001912. Prophylactic antibiotics for cystic fibrosis; Smyth A et al.; BACKGROUND: Cystic fibrosis blocks the airways with mucus and causes frequent respiratory infections . This leads to inflammation and lung disease, which frequently causes death from breathing failure . People with cystic fibrosis are sometimes given antibiotics regularly, in an attempt to prevent infections . However, antibiotics also have adverse effects, and longterm use might lead to the development of infections that can no longer be cleared by antibiotics . The review found some evidence from trials that preventive antibiotics for babies continued to two years may have some benefits . However, there is no evidence of the effects on adults or of longterm use . OBJECTIVES: We aimed to compare continuous oral antibiotic prophylaxis with no prophylaxis (short courses of oral antibiotics given as clinically indicated) in patients with cystic fibrosis . This review considers both the effectiveness of prophylaxis (bacteria isolated from the respiratory tract, requirement for additional antibiotic treatment, lung function, survival) and the adverse effects . SEARCH STRATEGY: The Cochrane Cystic Fibrosis and Genetic Disorders Group clinical trials register was used . This comprises references identified from a comprehensive search of electronic databases, as well as hand searching relevant journals and conference abstracts . Companies manufacturing anti-staphylococcal antibiotics were also approached for unpublished data . Date of the most recent search of the Group's specialised register: February 2001 . SELECTION CRITERIA: All randomised or pseudo-randomised trials where continuous oral prophylactic antibiotics, given for a period of at least one year, were compared to intermittent antibiotic therapy given "as required." Cystic fibrosis patients of any disease severity were considered . DATA COLLECTION AND ANALYSIS: Trials were assessed for eligibility, methodological quality and data extraction by the reviewers . The following outcomes were assessed: lung function; nutrition (weight standard deviation score); survival; requirement for additional antibiotic treatment; isolates of pathogens from the respiratory tract; occurrence of adverse reactions to prophylactic antibiotics . MAIN RESULTS: Three studies, totaling 177 patients aged 0-7 years on enrollment, were suitable for inclusion in the review . A reduced prevalence of Staphylococcus aureus in the respiratory secretions was seen in children receiving anti-staphylococcal antibiotic prophylaxis, although no effect was seen on other common pathogens . One eligible study showed a shorter duration of hospital admissions in the second year of life, in patients receiving prophylaxis . No effect on infant lung function has been shown after one year of prophylactic treatment . Data are not available on adverse effects of the interventions . There was a trend towards a lower cumulative isolation rate of Pseudomonas aeruginosa in the prophylaxis group, after three years . However, as the duration of the studies reviewed has been of three years or less, conclusions cannot be drawn about the long term effects of prophylaxis on acquisition of P . aeruginosa and survival . REVIEWER'S CONCLUSIONS: Anti-staphylococcal antibiotic prophylaxis may be of benefit when commenced early in infancy and continued up to three years of age . There is insufficient evidence from this review to say whether use in older children, or adults, or for periods of over three years is beneficial. J Biochem (Tokyo), 2001 Nov, 130(5), 711 - 7 nhaG Na(+)/H(+) antiporter gene of Bacillus subtilis ATCC9372, which is missing in the complete genome sequence of strain 168, and properties of the antiporter; Gouda T et al.; We cloned a gene which enabled Escherichia coli mutant host cells lacking all of the major Na(+)/H(+) antiporters to grow in the presence of 0.2 M NaCl from chromosomal DNA of Bacillus subtilis ATCC9372 . An Na(+)/H(+) antiport activity was observed with membrane vesicles prepared from E . coli cells possessing the cloned gene, but not with vesicles from the host cells . Lithium ion was also a substrate for the antiporter . We sequenced the cloned DNA and found one open reading frame (designated nhaG) preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence . The deduced amino acid sequence of NhaG suggested that it consisted of 524 residues and that the calculated molecular mass was 58.1 kDa . None of the bacterial Na(+)/H(+) antiporters so far reported, except NhaP of Pseudomonas aeruginosa and SynNhaP (NhaS1) of Synechocystis sp., showed significant sequence similarity with the NhaG . However, the NhaP, the SynNhaP, animal NHEs (Na(+)/H(+) exchangers), and some hypothetical Na(+)/H(+) antiporters of several organisms showed significant sequence similarities with the NhaG . Interestingly, the entire DNA region corresponding to the nhaG gene is missing in the reported complete genome sequence of B . subtilis strain 168 . We detected a band that hybridized with the nhaG DNA in chromosomal DNA from B . subtilis ATCC9372 but not with that from strain 168 . The missing DNA region (1,774 base pairs) is sandwiched by two identical sequences, TTTTCTT. Acta Otorhinolaryngol Belg, 2001, 55(3), 203 - 5 An unusual case of recurrent tonsillitis due to Pseudomonas aeruginosa; Danielides V et al.; An unusual case of recurrent tonsillitis due to pseudomonas aeruginosa . Pseudomonas (P.) aeruginosa in the head and neck region of an immunocompetent patient is mainly seen in ear infections, and sometimes in sinusitis . P . aeruginosa is an occasional finding in tonsil smears as part of normal microbial flora, but it rarely produces suppurative tonsil infection . We report a case of a previously healthy young female with recurrent episodes of tonsillitis due to P . aeruginosa infection . Although the patient received complete regimens of antibiotics (orally and intravenously) repeatedly, definitive eradication was only achieved after tonsillectomy. Environ Microbiol, 2001 Sep, 3(9), 561 - 9 Bioavailability of solid and non-aqueous phase liquid (NAPL)-dissolved phenanthrene to the biosurfactant-producing bacterium Pseudomonas aeruginosa 19SJ; Garcia-Junco M et al.; The biodegradation of phenanthrene by the biosurfactant-producing strain Pseudomonas aeruginosa 19SJ was investigated in experiments with the compound present either as crystals or dissolved in non-aqueous phase liquids (NAPLs) . Growth on solid phenanthrene exhibited an initial phase not limited by dissolution rate and a subsequent, carbon-limited phase caused by exhaustion of the carbon source . Rhamnolipid biosurfactants were produced from solid phenanthrene and appeared in solution and particulate material (cells and phenanthrene crystals) . During the carbon-limited phase, the concentration of rhamnolipids detected in culture exceeded the critical micelle concentration (CMC) determined with purified rhamnolipids . The biosurfactants caused a significant increase in dissolution rate and pseudosolubility of phenanthrene, but only at concentrations above the CMC . Externally added rhamnolipids at a concentration higher than the CMC increased the biodegradation rate of solid phenanthrene . Mineralization curves of low concentrations of phenanthrene initially dissolved in two NAPLs {2,2,4,4,6,8,8-heptamethylnonane and di(2-ethylhexyl)phthalate} were S-shaped, although no growth was observed in the population of suspended bacteria . Biosurfactants were not detected in solution under these conditions . The observed mineralization was attributed not only to suspended bacteria, but also to bacterial populations growing at the NAPL-water interface, mineralizing the compound at higher rates than predicted by abiotic partitioning . We suggest that rhamnolipid production and attachment increased the bioavailability of phenanthrene, so promoting biodegradation activity. Arch Pediatr, 2001 Aug, 8 Suppl 3, 603 - 609 {Evaluation of diagnosis and follow-up in screened children with cystic fibrosis in Normandy}; Brouard J et al.; The neonatal screening programme in Normandy (France) allowed the formation of a homogenous cystic fibrosis (CF) cohort of 150 children diagnosed between 1980 and 1997 . At the time of this retrospective study, 11 were deceased, out of which nine had meconium ileus (eight deaths after surgery, one at 5 years of age) . Sixty children born between 1980 and 1993 in the Basse-Normandie region were followed up during a mean 80 months following similar protocols . The mean age at diagnosis was 41 days (SD = 27 d) for infants without meconium ileus . The occurrence of Pseudomonas aeruginosa (P . aeruginosa) infection and chronic colonization was studied using a monovariate followed by a multivariate analysis including the following variables: sex; meconium ileus; anthropometric data at birth and at diagnosis; pancreatic insufficiency; radiological data (Brasfield score); microbiology data at diagnosis; and genetic data . P . aeruginosa infection appeared earlier in children with pancreatic insufficiency (OR = 2.2; p < 0.05) or with radiological abnormalities (Brasfield score < 21) at diagnosis (OR = 3.9; p < 0.05) . Meconium ileus (OR = 5.3; p < 0.01), pancreatic insufficiency (OR = 3.8; p < 0.01) and Brasfield score < 21 at diagnosis (OR = 5.6; p < 0.001) were prognosis factors for early chronic P . aeruginosa colonization . In CF children without meconium ileus, the major risk factor found through multivariate analysis for earlier infection and for earlier chronic colonization by P . aeruginosa was a diagnosis delay > 40 days (respectively OR = 4.6; p < 0.001 and OR = 10.4; p < 0.005) . These results must be compared with the lower Brasfield score at diagnosis in infants diagnosed after 40 days of life (p < 0.01). J Clin Microbiol, 2001 Nov, 39(11), 3976 - 81 Heterogeneity of Pseudomonas aeruginosa in Brazilian cystic fibrosis patients; Silbert S et al.; The aim of this study was to assess the diversity and genomic variability of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients being treated at a university hospital in Brazil . Ninety-seven isolates of P . aeruginosa from 43 CF patients were characterized by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE) and tested for susceptibility to 20 antimicrobial agents by broth microdilution . It was possible to evaluate single isolates from 20 patients and multiple isolates (two to seven) from 23 patients collected during a 22-month period . Among all of the unrelated patients, we detected only one pair of patients sharing a common strain . Among the 77 isolates from 23 patients who had multiple isolates analyzed, we identified 37 major types by PFGE, and five different colonization patterns were recognized . The isolates were susceptible to several antimicrobial agents, although consecutive isolates from the same patient may display differences in their susceptibilities . Mucoid isolates were more resistant (P < 0.001) than nonmucoid isolates to five antibiotics . Our results indicate that CF patients remain colonized by more than one strain of P . aeruginosa for long periods of time . In addition, the finding of several different genotypes in the same patient suggests that the colonizing strain may occasionally be replaced. J Biol Chem, 2002 Jan 4, 277(1), 424 - 31 Epub 2001 Oct 25. Tumor necrosis factor alpha increases the expression of glycosyltransferases and sulfotransferases responsible for the biosynthesis of sialylated and/or sulfated Lewis x epitopes in the human bronchial mucosa; Delmotte P et al.; There is increasing evidence that inflammation may affect glycosylation and sulfation of various glycoproteins . The present study reports the effect of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, on the glycosyl- and sulfotransferases of the human bronchial mucosa responsible for the biosynthesis of Lewis x epitope and of its sialylated and/or sulfated derivatives, which are expressed in human bronchial mucins . Fragments of macroscopically normal human bronchial mucosa were exposed to TNF-alpha at a concentration of 20 ng/ml . TNF-alpha was shown to increase alpha1,3-fucosyltransferase activity as well as expression of the two alpha1,3-fucosyltransferase genes expressed in the human airway, FUT3 and FUT4 . It had no influence on alpha1,2-fucosyltransferase activity or FUT2 expression . It also increased alpha2,3-sialyltransferase activity and the expression of ST3Gal-III and, more importantly, ST3Gal-IV and both N-acetylglucosamine 6-O-sulfotransferase and galactose 3-O-sulfotransferase . These results are consistent with the observation of oversialylation and increased expression sialyl-Lewis x epitopes on human airway mucins secreted by patients with severe lung infection such as those with cystic fibrosis, whose airways are colonized by Pseudomonas aeruginosa . However, other cytokines may also be involved in this process. J Antimicrob Chemother, 2001 Nov, 48(5), 717 - 21 OXA-35 is an OXA-10-related beta-lactamase from Pseudomonas aeruginosa; Aubert D et al.; Pseudomonas aeruginosa clinical isolate PA35 is resistant to amino- and ureido-penicillins, has intermediate susceptibility to cefsulodin, cefepime and aztreonam, and is susceptible to imipenem and ceftazidime . Cloning and sequencing revealed a new beta-lactamase variant, OXA-35, sharing 96% amino acid identity with OXA-10 . OXA-35 displays a restricted-substrate hydrolysis profile with improved hydrolysis of amoxicillin and cloxacillin compared with OXA-10 . OXA-35 differs from derivatives OXA-19 and OXA-28 by one amino acid substitution and may be a progenitor of these OXA-13-like extended-spectrum beta-lactamases. J Antimicrob Chemother, 2001 Nov, 48(5), 627 - 30 An integron-associated beta-lactamase (IBC-2) from Pseudomonas aeruginosa is a variant of the extended-spectrum beta-lactamase IBC-1; Mavroidi A et al.; An extended-spectrum beta-lactamase, IBC-2, produced by a clinical strain of Pseudomonas aeruginosa, was characterized . bla(IBC-2) was found, as a gene cassette, to be the sole gene within the variable region of a class 1 integron probably located in the chromosome . IBC-2 is a variant of IBC-1 and GES-1, differing by one amino acid from each of these beta-lactamases . When expressed in Escherichia coli, IBC-2 was observed to confer resistance to ceftazidime and decreased susceptibility to other oxyimino-beta-lactams. Appl Environ Microbiol, 2001 Nov, 67(11), 5254 - 60 Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation; Poirier Y et al.; Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads . Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase . The PHAC1 gene was put under the control of the promoter of the catalase A gene . PHA synthase expression and PHA accumulation were found in recombinant S . cerevisiae growing in media containing fatty acids . PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid . The maximum amount of PHA accumulated was 0.45% of the dry weight . Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles . Together, these data show that S . cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media . S . cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants. Fitoterapia, 2001 Nov, 72(7), 829 - 31 Antibacterial activity of Salvia tomentosa essential oil; Haznedaroglu MZ et al.; The essential oil of Salvia tomentosa aerial parts, consisting of 1,8-cineol (17%), beta-caryophyllene (11%), cyclofenchene (10%) and delta-cadinene (6%), was screened for its antimicrobial activity . The essential oil remarkably inhibited the growth of tested Gram-positive and Gram-negative bacteria except for Pseudomonas aeruginosa. Inorg Chem, 1999 Feb 8, 38(3), 433 - 438 X-ray Absorption Spectra of the Oxidized and Reduced Forms of C112D Azurin from Pseudomonas aeruginosa; DeBeer S et al.; The oxidized and reduced forms of a mutant of Pseudomonas aeruginosa azurin, in which the Cys112 has been replaced by an aspartate, have been studied by X-ray absorption spectroscopy . It is well established that the characteristic approximately 600 nm absorption feature of blue copper proteins is due to the S(Cys112) 3ppi --> Cu 3d(x)()()2(-)(y)()()2 charge-transfer transition . While other mutagenesis studies have involved the creation of an artificial blue copper site, the present work involves a mutant in which the native blue copper site has been destroyed, thus serving as a direct probe of the importance of the copper-thiolate bond to the spectroscopy, active site structure, and electron-transfer function of azurin . Of particular interest is the dramatic decrease in electron-transfer rates, both electron self-exchange (k(ese) approximately 10(5) M(-)(1) s(-)(1) wild-type azurin vs k(ese) approximately 20 M(-)(1) s(-)(1) C112D azurin) and intramolecular electron transfer to ruthenium-labeled sites (k(et) approximately 10(6) s(-)(1) wild-type azurin vs k(et) </= 10(3) s(-)(1) C112D azurin), which is observed in the mutant . These changes may be a reflection of significant differences in electronic coupling into the protein matrix (H(AB)) and/or in the reorganization energy (lambda) . These effects can be probed by the use of Cu K-edge X-ray absorption spectroscopy, the results of which indicate both a decrease in the covalency of the active site and an expansion of approximately 0.2 A in the Cu coordination sphere trigonal plane upon reduction of the C112D mutant. J Bacteriol, 2001 Nov, 183(22), 6676 - 83 The global posttranscriptional regulator RsmA modulates production of virulence determinants and N-acylhomoserine lactones in Pseudomonas aeruginosa; Pessi G et al.; Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria . Here we report the isolation of a Pseudomonas aeruginosa gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites) . Overexpression of rsmA resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin . While inactivation of rsmA in P . aeruginosa had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase . The influence of RsmA on N-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and N-butanoylhomoserine lactone (C4-HSL) produced by the rsmA mutant and the rsmA-overexpressing strain . RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using lasI and rhlI translational fusions . These data also highlighted the temporal expression control of the lasI gene, which was induced much earlier and to a higher level during the exponential growth phase in an rsmA mutant . To investigate whether RsmA modulates HCN production solely via quorum-sensing control, hcn translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (hcnABC) . RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the hcnA ribosome-binding site . This suggests that, in P . aeruginosa, RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry. J Bacteriol, 2001 Nov, 183(22), 6636 - 44 Interaction of the antiactivator FleN with the transcriptional activator FleQ regulates flagellar number in Pseudomonas aeruginosa; Dasgupta N et al.; Flagellar number in Pseudomonas aeruginosa is controlled by FleN, a putative ATP/GTP binding protein . Disruption of fleN results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect . We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily . In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system . Mutagenesis of the putative ATP/GTP binding motif in FleN(24K-->Q) and truncation of FleN at either the N or C terminus abrogates this interaction . FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator. J Bacteriol, 2001 Nov, 183(22), 6517 - 24 Molecular characterization and regulation of the aguBA operon, responsible for agmatine utilization in Pseudomonas aeruginosa PAO1; Nakada Y et al.; Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB) . The aguBA and adjacent aguR genes were cloned and characterized . The predicted AguB protein (M(r) 32,759; 292 amino acids) displayed sequence similarity (< or =60% identity) to enzymes of the beta-alanine synthase/nitrilase family . While the deduced AguA protein (M(r) 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis . The aguR gene encoded a putative regulatory protein (M(r) 24,424; 221 amino acids) of the TetR protein family . Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon . The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping . Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background . Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function. Eur J Med Chem, 2001 Sep, 36(9), 705 - 17 Synthesis and antibacterial study of unsaturated Mannich ketones; Lorand T et al.; Several Mannich ketones of 2-arylmethylenecycloalkanones were synthesised using the classical acid-catalysed Mannich reaction . Antibacterial activity of these new water-soluble compounds was reported against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus saprophyticus, Micrococcus luteus and Bacillus subtilis standard strains . Human cell line cytotoxicity of our new compounds was evaluated against HeLa cell lines . Some compounds showed low cytotoxicity (41.52 nM mL(-1) for 14 and 46.60 nM mL(-1) for 18) and proved to be efficient antibacterial agents against the Gram-positive strains . Minimum inhibitory concentrations varied from 1.56 to 100 microg mL(-1) . The mechanism of action was examined, too. Inorg Chem, 1997 Sep 24, 36(20), 4567 - 4570 Paramagnetic NMR Spectroscopy of Cobalt(II) and Copper(II) Derivatives of Pseudomonas aeruginosa His46Asp Azurin; Vila AJ et al.; NMR spectra of paramagnetic Co(II) and Cu(II) derivatives of Pseudomonas aeruginosa His46Asp azurin have been investigated . In each derivative, assignment of hyperfine-shifted resonances outside the diamagnetic envelope of spectra recorded at 200 and 500 MHz confirms that the Asp carboxylate is coordinated to the paramagnetic metal center . The reduced paramagnetic shifts of the Cys112 proton resonances in Cu(II) and Co(II) His46Asp azurins compared to those of the corresponding wild type proteins indicate that metal-S(Cys) bonding is weakened in this mutant . The downfield shifts of the gamma-CH(2) of Met121 suggest a stronger interaction between the metal and the Met thioether group than is present in the wild type protein . Molecular modeling of the metal site structure indicates a distorted tetrahedral geometry with Asp46 (monodentate carboxylate), Cys112, and His117 equatorial ligands . In this structure, the metal ion is shifted 0.3 A out of the O(Asp)S(Cys)N(His) trigonal plane toward Met121. Ann Otol Rhinol Laryngol, 2001 Oct, 110(10), 917 - 21 Effect of inhibitor of tumor necrosis factor-alpha on experimental otitis media with effusion; Jeon EJ et al.; Tumor necrosis factor (TNF)-alpha is important in the pathogenesis of otitis media with effusion (OME) . The purpose of this study was to determine the effect of TNF-alpha antagonist on the outcome of lipopolysaccharide (LPS)-induced OME in rats . Otitis media was induced by injecting Pseudomonas aeruginosa LPS transtympanically . Another (combination) group was pretreated with TNF-alpha antagonist, soluble TNF receptor type I (sTNF RI), before transtympanic injection of LPS . Saline and phosphate-buffered saline solutions were used as controls . Twelve hours after the transtympanic injection, otoscopic examination and aspiration of middle ear effusion (MEE) were done . The temporal bones in each group were examined histopathologically, and the vascular permeability of the middle ear mucosa was measured by the Evans blue vital dye technique . In the LPS and combination groups, MEE developed in 90% and 0% of ears, respectively . The combination group showed less inflammation, less mucosal thickening, and significantly decreased vascular permeability as compared to the LPS group . Transtympanic administration of sTNF RI appears to suppress the development of LPS-induced OME . This study suggests that TNF-alpha antagonist, along with antibiotics, may have an adjunctive role in the future treatment of MEE. Pathol Biol (Paris), 2001 Sep, 49(7), 534 - 9 {Survey of the antibiotic sensitivity of Pseudomonas aeruginosa in France and the distribution of beta-lactam resistance mechanisms: the GERPB 1999 study}; Cavallo JD et al.; A prospective survey was carried out in october 1999 in 15 french teaching hospitals . Average susceptibility rates, determined by minimal inhibitory concentrations, for the 738 non-repetitive strains of P . aeruginosa isolated were: ticarcillin, 58%, ticarcillin + clavulanic acid, 56%, piperacillin, 73%, piperacillin + tazobactam, 82%, ceftazidime, 76%, cefepime, 53%, cefpirome, 36%, aztreonam, 58%, imipenem, 81%, amikacin, 62%, tobramycine, 71% and, ciprofloxacin, 60% . Among the 75% serotypable strains, the most frequent serotypes were: O:6 (15.3%), O:11 (14.5%), O:1 (10.4%), O:3 (7.9%), O:4 (6.1%) and O:12 (6.1%) . The serotype O:12 was the most resistant to antibiotics . Forty-two percent of the strains were resistant or presented an intermediate susceptibility to ticarcillin . Mechanisms were as follow: 14.5% non enzymatic mechanism, 12.5% overproduction of the constitutive cephalosporinase, 7.1% transferable betalactamase and, 6.9% combination of these mechanisms . Among the 67 transferable betalactamases: 48 (71.6%) were PSE-1, 12 (19.4%) TEM-2 and 6 (7.5%) oxacillinases . One extended spectrum betalactamase was characterized . Among the cephalosporines tested, cefepime was less affected by the overproduction of constitutive cephalosporinase . Ceftazidime, remained the best cephalosporin except against the strains overexpressing the chromosomal type 1 beta-lactamase . Resistance to tobramycin was mainly due to enzymatic mechanisms with a high level of resistance . Decreased susceptibility was more frequent for amikacin than for tobramycin . This was probably related with non enzymatic mechanisms. Bioorg Khim, 2001 Sep-Oct, 27(5), 323 - 46 {Structure and functions of bacterial proteinase precursors}; Serkina AV et al.; The data on the precursors of bacterial proteases were generalized . The structure and special features of processing of the precursors of bacillary subtilisins, the alpha-lytic protease from Lysobacter enzymogenes and the related chymotrypsin-like proteases from Streptomyces griseus, and the metalloproteases from bacilli and Pseudomonas aeruginosa were discussed . The approaches to producing the precursors and the protease propeptides and to in vitro characterizing them were particularly analyzed . The following physiological functions of the propeptides within the protease precursors were considered probable: (a) inhibition of the proteases to protect the host cells from the proteolytic damage; (b) participation in the folding of the mature enzyme; and (c) providing for the protease interaction with the bacterial cell surveillance mechanisms, including protease translocation through the cell wall. Hybridoma, 2001 Aug, 20(4), 273 - 9 A monoclonal antibody specific for Pseudomonas aeruginosa amidase; Novo C et al.; Amidase from Pseudomonas aeruginosa was purified by anionic exchange chromatography and used to immunise female Balb/c mice . Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner . A selected IgM subclass MAb was purified from in vitro hybridoma cell line supernatant by a two-step anionic exchange chromatography . The MAb was specific for amidase from P . aeruginosa as determined by Western blotting and recognized the native and denatured forms of the enzyme. Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 731 - 5 Characterization of the lipA gene encoding the major lipase from Pseudomonas aeruginosa strain IGB83; Martinez A et al.; The lipases produced by Pseudomonas have a wide range of potential biotechnological applications . Pseudomonas aeruginosa IGB83 was isolated as a highly lipolytic strain which produced a thermotolerant and alkaline lipase . In the present work, we have characterized the P . aeruginosa IGB83 gene (lipA) encoding this enzyme . We describe the construction of a lipA mutant and report on the effect of two carbon sources on lipase expression. Antimicrob Agents Chemother, 2001 Nov, 45(11), 3256 - 61 Congeners of SMAP29 kill ovine pathogens and induce ultrastructural damage in bacterial cells; Kalfa VC et al.; SMAP29, an ovine cathelicidin, was systematically altered to create a family of 23 related peptides for MIC and minimum bactericidal concentration determinations . SMAP28, SMAP29, and a derivative of SMAP29 called ovispirin were all antimicrobial . However, many congeners of SMAP29 and ovispirin were not as active as the parent molecules . With immunoelectron microscopy, SMAP29 was seen on membranes and within the cytoplasm of Pseudomonas aeruginosa PAO1. Burns, 2001 Nov, 27(7), 723 - 30 Revised estimates of mortality from burns in the last 20 years at the Birmingham Burns Centre; Rashid A et al.; The Birmingham Burns Centre has been regularly presenting its mortality estimates since its pioneering work in 1949 on the use of probit analysis . The last of these estimates that showed a significant improvement in survival was presented in 1971 . This improvement was attributed to the introduction of topical 0.5% silver nitrate against Pseudomonas aeruginosa . In the last 20 years, several changes in management of burns have taken place following a better knowledge of its pathophysiology . This study shows our experience from the last 20 years by comparing mortality estimates between two successive 10-year periods i.e., 1979-1988 and 1989-1998 . We used probit analysis for deriving lethal area 50 (LA 50) for various age groups . The comparison showed that the mortality curves between the two periods were identical suggesting no improvement in the chances of survival . Since the mortalities were so similar the data were combined . The LA 50s derived from this combined data when compared with our earlier series from 1965 to 1970 also did not show a significant change in mortality . We conclude that in our experience the chances of dying for a given severity of injury have not changed significantly for more than 20 years. Rev Med Interne, 2001 Sep, 22(9), 877 - 80 {Ecthyma gangrenosum caused by Pseudomonas aeruginosa without septicemia in a neutropenic patient}; Versapuech J et al.; INTRODUCTION: Ecthyma gangrenosum is a rare skin infection caused by gram negative bacteria . It involves immunocompromised patients, especially neutropenic patients, and can be easily diagnosed . EXEGESIS: We report a case of ecthyma gangrenosum without septicemia due to Pseudomonas aeruginosa in a myelodysplastic patient with severe neutropenia . Granulocyte growth factors adjunction was necessary in combination to antibiotics to obtain complete healing . CONCLUSION: In neutropenic patient, ecthyma gangrenosum due to Pseudomonas aeruginosa should be rapidly diagnosed to avoid septicemic complications . In the case of antibiotic treatment failure, granulocyte growth factors may be added . Frequent Pseudomonas aeruginosa infections justify bacteriologic survey to look for hospital contamination. J Pediatr, 2001 Oct, 139(4), 572 - 7 Serum and lower respiratory tract drug concentrations after tobramycin inhalation in young children with cystic fibrosis; Rosenfeld M et al.; OBJECTIVES: To assess the serum and lower respiratory tract tobramycin concentrations (C(T)) produced by a single dose of tobramycin for inhalation delivered by a nebulizer and a compressor in patients with cystic fibrosis (CF) 6 months to 6 years of age . STUDY DESIGN: We performed a dose escalation study of serum C(T) measured before and 0.5, 1, 2, and 4 hours after a single dose of inhaled tobramycin, either 180 mg (10 patients) or 300 mg (19 patients) . In a separate group of 12 patients, epithelial lining fluid (ELF) C(T) was measured by bronchoalveolar lavage 30 to 45 minutes after a 300-mg dose . RESULTS: A 180-mg dose of inhaled tobramycin produced a mean peak serum C(T) of 0.5 microg/mL (SD 0.4; range, <0.2 to 1.4 microg/mL) . A 300-mg dose produced a mean peak serum C(T) of 0.6 microg/mL (SD 0.5; range, <0.2 to 1.2 microg/mL) . These peak values are well below the accepted maximum trough concentration with parenteral dosing (2 microg/mL) . The target ELF C(T) was 20 microg/mL, 10-fold greater than the minimal inhibitory concentration for most Pseudomonas aeruginosa isolates from very young patients with CF (2 microg/mL) . Mean ELF C(T) was 90 microg/mL (SD 54; range, 16 to 204 microg/mL) and exceeded the target concentration in 11 patients . CONCLUSION: In patients with CF ages 6 months to 6 years, a single 300-mg dose of inhaled tobramycin appears to produce safe peak serum concentrations and drug concentrations in the bactericidal range in the lower respiratory tract. Infect Immun, 2001 Nov, 69(11), 6962 - 9 Dual-function vaccine for Pseudomonas aeruginosa: characterization of chimeric exotoxin A-pilin protein; Hertle R et al.; Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis patients . Strategies to prevent colonization by this bacterium and/or neutralize its virulence factors are clearly needed . Here we characterize a dual-function vaccine designed to generate antibodies to reduce bacterial adherence and to neutralize the cytotoxic activity of exotoxin A . To construct the vaccine, key sequences from type IV pilin were inserted into a vector encoding a nontoxic (active-site deletion) version of exotoxin A . The chimeric protein, termed PE64Delta553pil, was expressed in Escherichia coli, refolded to a near-native conformation, and then characterized by various biochemical and immunological assays . PE64Delta553pil bound specifically to asialo-GM1, and, when injected into rabbits, produced antibodies that reduced bacterial adherence and neutralized the cell-killing activity of exotoxin A . Results support further evaluation of this chimeric protein as a vaccine to prevent Pseudomonas colonization in susceptible individuals. Am J Physiol Lung Cell Mol Physiol, 2001 Nov, 281(5), L1240 - 7 Direct and indirect bacterial killing functions of neutrophil defensins in lung explants; Porro GA et al.; Studies of the antimicrobial activity of neutrophil defensins have mostly been carried out in microbiological media, and their effects on the host defense in physiological conditions are unclear . We examined 1) the antibacterial activity of defensins in physiological media with and without lung tissue present, 2) the effect of defensins on hydrogen peroxide (H(2)O(2)) production by lung tissue that had been exposed to bacteria, and 3) the effect of diphenyleneiodonium (DPI), an inhibitor of reactive oxygen species formation, on the antibacterial activity of defensins in the presence of lung tissue . Defensins were incubated with Escherichia coli or Pseudomonas aeruginosa in the absence or presence of primary cultured mouse lung explants . Defensins reduced bacterial counts by approximately 65-fold and approximately 25-fold, respectively, at 48 h; bacterial counts were further decreased by approximately 600-fold and approximately 12,000-fold, respectively, in the presence of lung tissue . Defensins induced H(2)O(2) production by lung tissue, and the rate of killing of E . coli by defensins was reduced by approximately 2,500-fold in the presence of 10 microM DPI . We conclude that defensins exert a significant antimicrobial effect under physiological conditions and that this effect is enhanced in the presence of lung tissue by a mechanism that involves the production of reactive oxygen species. J Gastroenterol Hepatol, 2001 Sep, 16(9), 1055 - 9 Incidence, spectrum and antibiotic sensitivity pattern of bacterial infections among patients with acute pancreatitis; Garg PK et al.; BACKGROUND AND AIM: Secondary infection of pancreatic necrotic tissue and peripancreatic fluid is a serious complication of acute pancreatitis resulting in significant morbidity and mortality . The aim of this study was to find out the spectrum of bacterial infections, and their antibiotic sensitivity pattern in patients with acute pancreatitis . METHODS: All consecutive patients with acute pancreatitis were studied prospectively . Detailed investigations were carried out to identify bacterial infections and their antibiotic sensitivities in patients with suspected infection . These investigations included cultures of various body fluids, throat swabs, indwelling cannula and catheter tips . Pancreatic tissue was obtained by using needle aspiration or at surgery for Gram's stain, culture and sensitivity . All cultures were repeated until the presence of infection was confirmed or excluded . RESULTS: A total of 169 patients with acute pancreatitis were studied during the period between January 1997 and June 2000 (mean age 41.3 years; 116 males and 53 females) . Of the 169 patients, 63 had infections at various sites . A total of 80 cultures were positive, and 12 different bacterial isolates were cultured from samples taken from these 63 patients . Polymicrobial infection was seen in 32% of patients . Twenty-four patients had a confirmed pancreatic infection . Blood cultures had a growth of organisms in 19 patients, with evidence of ongoing or worsening pancreatitis, thus raising a strong suspicion of infected necrosis in them . The commonest organisms were Escherichia coli from 20 cultures and Pseudomonas aeruginosa from 18 cultures . The antibiotic sensitivity pattern showed that most bacteria were sensitive to third generation cephalosporins and quinolones; notably among them were cefotaxime, ceftazidime, and ciprofloxacin . CONCLUSION: Bacterial infections were seen in 37% of patients with acute pancreatitis . The commonest organisms were Pseudomonas aeruginosa and Escherichia coli . Most bacterial isolates were sensitive to third generation cephalosporins and quinolones. Microbiol Immunol, 2001, 45(8), 579 - 90 Iron-Mediated regulation of alkaline proteinase production in Pseudomonas aeruginosa; Shigematsu T et al.; We analyzed the regulation by iron of alkaline proteinase (AP) production in Pseudomonas aeruginosa . Extracellular AP production was detected from the mid-logarithmic to the stationary phase by an antibody-based assay system, and was strongly repressed by iron in the medium . This repression was shown by Northern hybridization and primer extension to occur at the level of transcription . The primer extension analysis revealed that the start point of transcription of AP gene was the nucleotide position -84 from the start point of translation . Furthermore, we investigated whether this transcriptional repression involved PvdS protein . Using the mutant strain of pvdS, the alternative sigma factor gene revealed that the PvdS protein is required for the full expression of AP, and a previous study showed that expression of pvdS is also repressed by iron . Therefore, we thought that one mechanism of repression of AP production operated through reduction of the PvdS protein level . Purified AP decomposed the transferrin, and released iron from it . Purified AP added to the medium containing transferrin as the only iron source enhanced the growth of P . aeruginosa . Moreover, mutation in the AP gene decreased the growth rate in the medium containing the transferrin as the only iron source . These results clearly indicated that AP expression should occur in a free-iron-deficient environment and emphasized the importance of AP to iron acquisition in the infection site. Mol Med, 2001 Aug, 7(8), 523 - 34 Control of the proinflammatory state in cystic fibrosis lung epithelial cells by genes from the TNF-alphaR/NFkappaB pathway; Eidelman O et al.; BACKGROUND: Cystic fibrosis (CF) is the most common, lethal autosomal recessive disease affecting children in the United States and Europe . Extensive work is being performed to develop both gene and drug therapies . The principal mutation causing CF is in the CFTR gene ({Delta F508}CFTR) . This mutation causes the mutant protein to traffic poorly to the plasma membrane, and degrades CFTR chloride channel activity . CPX, a candidate drug for CF, binds to mutant CFTR and corrects the trafficking deficit . CPX also activates mutant CFTR chloride channel activity . CF airways are phenotypically inundated by inflammatory signals, primarily contributed by sustained secretion of the proinflammatory cytokine interleukin 8 (IL-8) from mutant CFTR airway epithelial cells . IL-8 production is controlled by genes from the TNF-alphaR/NFkappaB pathway, and it is possible that the CF phenotype is due to dysfunction of genes from this pathway . In addition, because drug therapy with CPX and gene therapy with CFTR have the same common endpoint of raising the levels of CFTR, we have hypothesized that either approach should have a common genomic endpoint . MATERIALS AND METHODS: To test this hypothesis, we studied IL-8 secretion and global gene expression in IB-3 CF lung epithelial cells . The cells were treated by either gene therapy with wild-type CFTR, or by pharmacotherapy with the CFTR-surrogate drug CPX . CF cells, treated with either CFTR or CPX, were also exposed to Pseudomonas aeruginosa, a common chronic pathogen in CF patients . cDNA microarrays were used to assess global gene expression under the different conditions . A novel bioinformatic algorithm (GENESAVER) was developed to identify genes whose expression paralleled secretion of IL-8 . RESULTS: We report here that IB3 CF cells secrete massive levels of IL-8 . However, both gene therapy with CFTR and drug therapy with CPX substantially suppress IL-8 secretion . Nonetheless, both gene and drug therapy allow the CF cells to respond with physiologic secretion of IL-8 when the cells are exposed to P . aeruginosa . Thus, neither CFTR nor CPX acts as a nonspecific suppressor of IL-8 secretion from CF cells . Consistently, pharmacogenomic analysis indicates that CF cells treated with CPX greatly resemble CF cells treated with CFTR by gene therapy . Additionally, the same result obtains in the presence of P . aeruginosa . Classical hierarchical cluster analysis, based on similarity of global gene expression, also supports this conclusion . The GENESAVER algorithm, using the IL-8 secretion level as a physiologic variable, identifies a subset of genes from the TNF-alphaR/NFkappaB pathway that is expressed in phase with IL-8 secretion from CF epithelial cells . Certain other genes, previously known to be positively associated with CF, also fall into this category . Identified genes known to code for known inhibitors are expressed inversely, out of phase with IL-8 secretion . CONCLUSIONS: Wild-type CFTR and CPX both suppress proinflammatory IL-8 secretion from CF epithelial cells . The mechanism, as defined by pharmacogenomic analysis, involves identified genes from the TNF-alphaR/NFkappaB pathway . The close relationship between IL-8 secretion and genes from the TNF-alphaR/NFkappaB pathway suggests that molecular or pharmaceutical targeting of these novel genes may have strategic use in the development of new therapies for CF . From the perspective of global gene expression, both gene and drug therapy have similar genomic consequences . This is the first example showing equivalence of gene and drug therapy in CF, and suggests that a gene therapy-defined endpoint may prove to be a powerful paradigm for CF drug discovery . Finally, because the GENESAVER algorithm is capable of isolating disease-relevant genes in a hypothesis-driven manner without recourse to any a priori knowledge about the system, this new algorithm may also prove useful in applications to other genetic diseases. J Bacteriol, 2001 Nov, 183(21), 6413 - 21 Bacterial two-hybrid analysis of interactions between region 4 of the sigma(70) subunit of RNA polymerase and the transcriptional regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa; Dove SL et al.; A number of transcriptional regulators mediate their effects through direct contact with the sigma(70) subunit of Escherichia coli RNA polymerase (RNAP) . In particular, several regulators have been shown to contact a C-terminal portion of sigma(70) that harbors conserved region 4 . This region of sigma contains a putative helix-turn-helix DNA-binding motif that contacts the -35 element of sigma(70)-dependent promoters directly . Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-sigma factor Rsd and the sigma(70) subunit of E . coli RNAP . Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of sigma(70) and also that amino acid substitution R596H, within region 4 of sigma(70), weakens this interaction . We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between sigma and two other regulators shown previously to contact region 4 of sigma(70) . We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression in Pseudomonas aeruginosa, can contact the C-terminal region of the sigma(70) subunit of RNAP from this organism . We found that amino acid substitution R600H in sigma(70) from P . aeruginosa, corresponding to the R596H substitution in E . coli sigma(70), specifically weakens the interaction between AlgQ and sigma(70) . Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of sigma(70) and probably regulate gene expression through this contact. J Bacteriol, 2001 Nov, 183(21), 6207 - 14 Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning; Gallagher LA et al.; In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans . Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P . aeruginosa that is responsible for killing of the nematode . Four lines of evidence support this conclusion . First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing . Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency . Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria . The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure . And fourth, a nematode mutant (egl-9) resistant to P . aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria . A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented . The action of cyanide helps account for the unusually broad host range of virulence of P . aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium. J Autoimmun, 2001 Sep, 17(2), 165 - 72 Autoantibodies to pancreatic hsp60 precede the development of glucose intolerance in patients with cystic fibrosis; Jensen P et al.; Persons expressing the genetic disease cystic fibrosis (CF) suffer from a high risk of developing impaired glucose tolerance and diabetes . The development of diabetes in CF has been attributed, in the past, to the destruction of pancreatic islets and their resident beta-cells secondary to the destruction of the surrounding tissue by mechanical clogging of the pancreatic exocrine ducts . However, the discovery that autoimmunity to the 60-kDa heat shock protein (hsp60) may cause type I diabetes in NOD mice raises the possibility that hsp60 autoimmunity may be involved in CF diabetes too; could the hyperimmunization to bacterial hsp60 characteristic of CF spread to self-hsp60 and hence to autoimmune diabetes? We now report that rising levels of IgG autoantibodies to hsp60 do indeed precede the appearance of glucose intolerance and diabetes in CF patients . We produced a recombinant human pancreatic hsp60 protein and investigated the IgG antibody response to hsp60 in prediabetic and non-diabetic patients with CF . To detect hsp60 autoantibodies in the presence of high levels of antibodies to bacterial hsp60, we absorbed test sera with the 60-kDa GroEL of Pseudomonas aeruginosa and used an immunostaining technique . Using this technique, 32 prediabetic CF patients were evaluated over a five-year period, three years, on the average, before the onset of glucose intolerance . We found that a significant increase in hsp60 autoantibody preceded impaired glucose tolerance (P=0.042, n=17), diabetes (P=0.011, n=15) and glucose intolerance (P=0.005, n=32) . As has been observed in NOD mice and in type I diabetic patients, the hsp60 autoantibodies decline at the outbreak of glucose intolerance in the CF patients . The association of CF diabetes with the rise and fall of hsp60 autoimmunity suggests that the pathogenesis of the diabetes may not be merely mechanical, but arise in the wake of bacterial hyperimmunisation . J Chemother, 2001 Aug, 13(4), 354 - 62 The role of nebulized antibiotics in treating serious respiratory infections; Cole PJ; The administration of a nebulized antibiotic in serious respiratory tract infections ensures high antibiotic concentrations at the site of infection, minimising systemic concentrations and their resultant risk of toxicity . Nebulized antibiotics have been used for the treatment of chronic infection with Pseudomonas aeruginosa, particularly in cystic fibrosis, but with variable clinical efficacy . Antibiotic delivery by nebulization is greatly influenced by the product formulation and the nebulizer . Use of intravenous formulations via a nebulizer can lead to exposure to potentially irritant or toxic additives and inappropriate pH or osmolality ranges, whilst the choice of nebulizer can greatly influence the drug deposition in the airway . Tobramycin Nebulizer Solution (TNS) is the first specific formulation for nebulization in cystic fibrosis using a designated nebulizer . The potential extrapolation of nebulized antibiotic therapy to other serious respiratory infections, in particular bronchiectasis and ventilator-associated pneumonia, is explored in this review. Chemotherapy, 2001, 47 Suppl 4, 39 - 46; discussion 53-4 Management of clinical failures in non-ICU patients with chronic obstructive pulmonary disease exacerbations; Habib MP; Clinical failure after initial treatment for exacerbations of chronic obstructive pulmonary disease (COPD) occurs in 10-25% of cases . Once the original diagnosis is confirmed, there is a need to optimise therapy, including introducing bronchodilators and corticosteroids . The use of aggressive antibiotic treatment is recommended for patients with risk factors (elderly, more than four exacerbations per year, underlying cardiopulmonary disease) and more severe disease . Fluoroquinolones are a good choice for those patients who failed initial therapy and who require antimicrobials, including those with simple exacerbations, complicated cases with comorbidity, or those with bronchiectasis . Consideration of less common pathogens, such as Pseudomonas aeruginosa infection, should also be considered . Bacteria usually associated with exacerbations are becoming increasingly resistant, and this needs to be considered when deciding on appropriate antibiotic treatment . Lancet, 2001 Sep 22, 358(9286), 983 - 4 Effect of inhaled tobramycin on early Pseudomonas aeruginosa colonisation in patients with cystic fibrosis; Ratjen F et al.; Early antibiotic treatment of airway colonisation with Pseudomonas aeruginosa can delay onset of chronic lung infection in patients with cystic fibrosis . Whether the pathogen is eradicated by this treatment is unclear . We successfully eradicated the organism in 14 of 15 patients with cystic fibrosis who had been colonised by P aeruginosa . Patients inhaled 80 mg tobramycin twice daily for 12 months . Eradication was confirmed by sequential respiratory cultures and serum antibody titres that were negative for P aeruginosa . Our antibiotic therapy regimen maintained pulmonary function at high levels. Infect Control Hosp Epidemiol, 2001 Jul, 22(7), 409 - 13 Nosocomial transmission of imipenem-resistant Pseudomonas aeruginosa following bronchoscopy associated with improper connection to the Steris System 1 processor; Sorin M et al.; OBJECTIVE: To assess nosocomial transmission of imipenem-resistant Pseudomonas aeruginosa (IRPA) following bronchoscopy during August through October 1998 . DESIGN: Traditional and molecular epidemiological investigation of a case series . SETTING: University-affiliated community hospital . PATIENTS: 18 patients with IRPA bronchial-wash isolates . INTERVENTIONS: We reviewed clinical data, performed environmental cultures and molecular analysis of all IRPA isolates, and observed disinfection of bronchoscopes . RESULTS: Of 18 patients who had IRPA isolated from bronchoscopic or postbronchoscopic specimens, 13 underwent bronchoscopy for possible malignancy or undiagnosed pulmonary infiltrates . Following bronchoscopy, 3 patients continued to have IRPA isolated from sputum and demonstrated clinical evidence of infection requiring specific antimicrobial therapy . The remaining 15 patients had no further IRPA isolated and remained clinically well 3 months following bronchoscopy . Pulsed-field gel electrophoresis revealed that all strains except one were >95% related . STERIS SYSTEM 1 had been implemented in July 1998 as an automatic endoscope reprocessor (AER) for all endoscopes and bronchoscopes . Inspection of bronchoscope sterilization cycles revealed incorrect connectors joining the bronchoscope suction channel to the STERIS SYSTEM 1 processor, obstructing peracetic acid flow through the bronchoscope lumen . No malfunction warning was received, and spore strips remained negative . CONCLUSIONS: The similarity of diverse connectors and limited training by the manufacturer regarding AER for bronchoscopes were the two factors responsible for the outbreak . Appropriate connections were implemented, and there was no further bronchoscope contamination . We suggest active surveillance of all bronchoscopy specimen cultures, standardization of connectors of various scopes and automated processors, and systematic education of staff by manufacturers with periodic on-site observation. Klin Padiatr, 2001 Sep-Oct, 213(5), 285 - 7 Successful treatment of Pseudomonas aeruginosa respiratory tract infection with a sugar solution--a case report on a lectin based therapeutic principle; von Bismarck P et al.; BACKGROUND: Airway infections with Pseudomonas aeruginosa often represent a life-threatening event in immuno-compromised patients or patients with Cystic Fibrosis . The adhesion of this bacterium to surfaces such as the airway epithelium is mediated by two lectins, sugar binding proteins . In addition to their adhesive properties, these lectins have been shown to stop human ciliary beating thus compromising the mucociliary clearance as an important non-specific defence mechanism of the airways . Inhibition of these lectins by their specific sugars galactose and fucose, respectively, could therefore be of benefit in the elimination therapy of P . aeruginosa . CASE REPORT: An infant suffering from P . aeruginosa airway infection after chemotherapy for neuroblastoma, which could not successfully be treated by antibiotics, was subjected to a series of additional galactose/fucose inhalations, which eliminated the germ as evidenced by microbiological testing . This is the first report suggesting the effectiveness of a lectin-based therapeutic principle in P . aeruginosa airway infection . CONCLUSION: The competitive inhibition of P . aeruginosa lectins by the lectin specific sugars galactose and fucose may overcome particular mechanisms of bacterial resistance in patients with P . aeruginosa airway infection . This underlying biochemical mechanism and the outcome of our patient suggest a clinical benefit of this novel therapeutic approach for immunocompromised patients or patients with cystic fibrosis suffering from infection with P . aeruginosa. J Antimicrob Chemother, 2001 Oct, 48(4), 553 - 5 In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy; Le Thomas I et al.; We report the emergence after 4 days of ciprofloxacin monotherapy of a double mutant of Pseudomonas aeruginosa overexpressing the multidrug efflux system MexAB-OprM and harbouring a mutation in the gyrB gene . Compared with its initial susceptible counterpart, this mutant exhibited a significant increase in resistance to most of the beta-lactam antibiotics tested (16 x MIC of ticarcillin) and to ciprofloxacin (128 x MIC) . Combined ceftazidime and amikacin therapy finally eradicated the resistant isolate and cured the patient of his infection . This case illustrates how strains of P . aeruginosa may develop high levels of fluoroquinolone resistance by combining efflux mechanisms and target alterations. J Antimicrob Chemother, 2001 Oct, 48(4), 527 - 34 Cefepime versus cefotaxime for empirical treatment of bacterial pneumonia in HIV-infected patients: an open, randomized trial; Cordero E et al.; An open, randomized, multicentre clinical trial was conducted to compare the efficacy and safety of cefepime 2 g iv bd (2 g tds daily in cases of Pseudomonas aeruginosa pneumonia) with cefotaxime 2 g iv tds, in the empirical treatment of bacterial pneumonia in HIV-infected patients . The primary end-point was effectiveness after 3-5 days of treatment, taking success to be when the study drug was continued during this period of time . Clinical and bacteriological responses at end of treatment (EOT) were also evaluated . Analyses of the intention-to-treat population (n = 160) and the as-per-protocol groups (n = 150) were carried out . Treatment gro