Clin Exp Immunol, 1997 Apr, 108(1), 88 - 94 The influence of allotypes on the IgG subclass response to chromosomal beta-lactamase of Pseudomonas aeruginosa in cystic fibrosis patients; Ciofu O et al.; Sera from 70 adult cystic fibrosis (CF) patients with chronic lung infection with Pseudomonas aeruginosa were typed for seven GM and two KM allotype determinants . IgG class and all four IgG subclasses of antibodies against chromosomal beta-lactamase of Ps . aeruginosa (a beta ab) were measured in all 70 CF patients in a cross-sectional study . The a beta ab IgG subclass response in sera collected during the first 11 years of chronic infection from 20 CF patients (10 patients with G3M*5 G1M*3/G3M*5 G1M*3 genotype and 10 patients with G3M*21 G1M*1/G3M*21 G1M*1 genotype) was analysed in a longitudinal study . Increased levels of IgG2 were associated with the presence of GM 23 allotype . IgG3 a beta ab levels were the lowest for subjects with the GM 1,2,3,17 23 5,21 and GM 1,3,17 21 phenotypes and the highest in subjects with GM 3,23,5 and GM 3,5 . No significant differences in IgG1 and IgG4 a beta ab levels were found between the different phenotypes . IgG1 a beta ab levels were higher in patients with KM*3/KM*3 genotype compared with patients with KM*3, *1 genotype . Patients with G3M*5 G1M*3/G3M*5 G1M*3 genotype had in both the cross-sectional and the longitudinal study higher IgG3 a beta ab, lower IgG4 a beta ab levels and poorer lung function than patients with G3M*21 G1M*1/ G3M*21 G1M*1 genotype . An influence of the allotypes on the clinical course of chronic lung infection with Ps . aeruginosa in patients with CF is suggested. Appl Environ Microbiol, 1997 Apr, 63(4), 1389 - 95 Isolation and characterization of an attenuated strain of Pseudomonas aeruginosa AC869, a 3,5-dichlorobenzoate degrader; Zhou X et al.; Pseudomonas aeruginosa AC869, a 3,5-dichlorobenzoate degrader, is a mouse pathogen and has a reported 50% lethal dose (LD50) of 1.05 x 10(7) CFU when given intranasally to C3H/HeJ mice (S.E . George, M.J . Kohan, M.I . Gilmour, M.S . Taylor, H.G . Brooks, J.P . Creason, and L.D . Claxton, Appl . Environ, Microbiol . 59:3585-3591, 1993) . AC869 was serotyped as O6 when grown in CD-1 mouse cecal and lung mucus but could not be assigned an O serotype when grown in Luria broth (LB) . After growth in mouse cecal mucus, a less virulent mutant, AC869-11, was isolated from AC869 by using bacteriophage E79, which adsorbs to the O side chain of lipopolysaccharide (LPS) . AC869-11 produced significantly less O antigen on its LPS than AC869 when grown in mouse lung and cecal mucus . The mutant also produced half the amount of exoenzyme S and 16-fold less extracellular protease than AC869 and was more sensitive than its parent to a number of antibiotics when grown either in LB or in mouse lung mucus . AC869-11 had ninefold higher LD50 than AC869 in CD-1 mice when administered intranasally . AC869-11 was found in the lungs, small intestine, cecum, and large intestine in numbers at least 100-fold below AC869, 3 h after intranasal exposure of mice to a sublethal dose of the two strains . Moreover, AC869-11 induced a decreased pulmonary inflammatory response relative to AC869 . In contrast to AC869, AC869-11 did not translocate to the mesenteric lymph nodes, liver, and spleen following a sublethal dose . Despite attenuation, AC869-11 grew as well as AC869 with 3,5-dichlorobenzoate as the sole carbon and energy source . However, although AC869-11 survived in 3,5-dichlorobenzoate-contaminated soil as well as AC869 for 1 week, it failed to survive as well thereafter . These results suggest the possibility that mutations that lead to pulmonary attenuation of P . aeruginosa in mice also lead to weakness in the environment, despite such mutants maintaining the ability to degrade toxic substances under laboratory conditions. Antimicrob Agents Chemother, 1997 Apr, 41(4), 823 - 6 Adaptive resistance of Pseudomonas aeruginosa induced by aminoglycosides and killing kinetics in a rabbit endocarditis model; Xiong YQ et al.; Adaptive resistance following the first exposure to aminoglycosides is a recently described in vitro phenomenon in Pseudomonas aeruginosa and other aerobic gram-negative bacilli . We investigated the in vivo relevance of adaptive resistance in P . aeruginosa following a single dose of amikacin in the experimental rabbit endocarditis model . Rabbits with P . aeruginosa endocarditis received either no therapy (control) or a single intravenous (i.v.) dose of amikacin (80 mg/kg of body weight) at 24 h postinfection, after which they were sacrificed at 5, 8, 12, 16, or 24 h postdose . Excised aortic vegetations were subsequently exposed ex vivo to amikacin at 2.5, 5, 10 or 20 times the MIC for 90 min . In vivo adaptive resistance was identified when amikacin-induced pseudomonal killing within excised aortic vegetations was less in animals receiving single-dose amikacin in vivo than in vegetations from control animals not receiving amikacin in vivo . Maximal adaptive resistance occurred between 8 and 16 h after the in vivo amikacin dose, with complete refractoriness to ex vivo killing by amikacin seen at 12 h postdose . By 24 h postdose, bacteria within excised vegetations had partially recovered their initial amikacin susceptibility . In a parallel treatment study, we demonstrated that amikacin given once daily (but not twice daily) at a total dose of 80 mg/kg i.v . for 1-day treatment significantly reduced pseudomonal densities within aortic vegetations versus those in untreated controls . When therapy was continued for 3 days with the same total daily dose (80 mg/kg/day), amikacin given once or twice daily significantly reduced intravegetation pseudomonal densities versus those in controls . However, amikacin given once daily was still more effective than the twice-daily regimen . These data confirm the induction of aminoglycoside adaptive resistance in vivo and further support the advantages of once-daily aminoglycoside dosing regimens in the treatment of serious pseudomonal infections. Antimicrob Agents Chemother, 1997 Apr, 41(4), 785 - 90 OXA-15, an extended-spectrum variant of OXA-2 beta-lactamase, isolated from a Pseudomonas aeruginosa strain; Danel F et al.; Pseudomonas aeruginosa AH, isolated in Ankara, Turkey, was highly resistant to ceftazidime (MIC, 128 microg/ml) and produced a beta-lactamase that gave a doublet of bands at pIs 8.7 and 8.9 . beta-Lactamase production was transferable to P . aeruginosa PU21 by conjugation and was determined by a ca . 450-kb plasmid, pMLH54 . The transconjugant and Escherichia coli transformed with the cloned gene showed increased resistance to ceftazidime (especially) and to cefpirome, ceftazidime, ceftriaxone, moxalactam, and aztreonam, but not to carbapenems . Resistance was not reversed by clavulanic acid or tazobactam . Sequencing revealed that the beta-lactamase responsible for this resistance was identical to OXA-2 except that glycine replaced aspartate at position 150 . Compared to OXA-2, the new enzyme, named OXA-15, had greater cephalosporinase activity, with increased relative hydrolysis rates for cephaloridine and cephalothin and, most dramatically, for ceftazidime . Cefotaxime and carbapenems remained stable to hydrolysis . Thus, as in the TEM, SHV, and OXA-10 (PSE-2) beta-lactamase families, a minor sequence change in OXA-2 gave a major extension of cephalosporinase activity and contingent resistance . The gene encoding the new beta-lactamase, bla(OXA-15), lay close to the highly conserved 3' end of an integron and had flanking sequences typical of an integron-associated gene cassette . Restriction mapping and partial sequence data indicated that pMLH54 carries an integron with three putative gene cassettes: bla(OXA-15) itself, aadB {coding aminoglycoside nucleotidyltransferase (2")-1a}, and an uncharacterized cassette. Antimicrob Agents Chemother, 1997 Apr, 41(4), 733 - 8 Pharmacokinetic-pharmacodynamic modeling of activity of ceftazidime during continuous and intermittent infusion; Mouton JW et al.; We developed and applied pharmacokinetic-pharmacodynamic (PK-PD) models to characterize in vitro bacterial rate of killing as a function of ceftazidime concentrations over time . For PK-PD modeling, data obtained during continuous and intermittent infusion of ceftazidime in Pseudomonas aeruginosa killing experiments with an in vitro pharmacokinetic model were used . The basic PK-PD model was a maximum-effect model which described the number of viable bacteria (N) as a function of the growth rate (lambda) and killing rate (epsilon) according to the equation dN/dt = {lambda - epsilon x {Cgamma(EC50gamma + Cgamma)}} N, where gamma is the Hill factor, C is the concentration of antibiotic, and EC50 is the concentration of antibiotic at which 50% of the maximum effect is obtained . Next, four different models with increasing complexity were analyzed by using the EDSIM program (MediWare, Groningen, The Netherlands) . These models incorporated either an adaptation rate factor and a maximum number of bacteria (Nmax) factor or combinations of the two parameters . In addition, a two-population model was evaluated . Model discrimination was by Akaike's information criterion . The experimental data were best described by the model which included an Nmax term and a rate term for adaptation for a period up to 36 h . The absolute values for maximal growth rate and killing rate in this model were different from those in the original experiment, but net growth rates were comparable . It is concluded that the derived models can describe bacterial growth and killing in the presence of antibiotic concentrations mimicking human pharmacokinetics . Application of these models will eventually provide us with parameters which can be used for further dosage optimization. J Bacteriol, 1997 Apr, 179(7), 2339 - 47 The kilE locus of promiscuous IncP alpha plasmid RK2 is required for stable maintenance in Pseudomonas aeruginosa; Wilson JW et al.; Eight coordinately regulated operons constitute the kor regulon of the IncP alpha plasmid RK2 . Three operons specify functions required for replication initiation, conjugative transfer, and control of gene expression . The functions of the other operons, including those of the four coregulated operons that compose the kilA, kilC, and kilE loci, have not been determined . Here, we present the first evidence that a kil determinant is involved in IncP plasmid maintenance . Elevation of KorC levels specifically to reduce the expression of the KorC-regulated kilC and kilE operons severely affected the maintenance of both the IncP alpha plasmid RK2lac and the IncP beta plasmid R751 in Pseudomonas aeruginosa but had little effect on plasmid maintenance in Escherichia coli . Precise deletion of the two kilE operons from RK2lac was achieved with the VEX mutagenesis system for large genomes . The resulting plasmid showed significant loss of stability in P . aeruginosa only . The defect could be complemented by reintroduction of kilE at a different position on the plasmid . The instability of the RK2lac delta kilE mutant did not result from a reduction in average plasmid copy number, reduced expression of kilC, decreased conjugative transfer, or loss of the korE regulator . We found that both the par and kilE loci are required for full stability of RK2lac in P . aeruginosa and that the par and kilE functions act independently . These results demonstrate a critical role for the kilE locus in the stable inheritance of RK2 in P . aeruginosa. J Bacteriol, 1997 Apr, 179(7), 2181 - 8 Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis; Chopade BA et al.; We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosine triphosphatase (GTPase) activity . The protein was purified as a 48-kDa polypeptide capable of binding and hydrolyzing GTP . The N-terminal sequence of the purified protein revealed its similarity to the Escherichia coli Ras-like protein (Era), and the protein cross-reacted with anti-Era antibodies . This protein was named Pseudomonas Ras-like protein (Pra) . Anti-Pra antibodies also cross-reacted with E . coli Era protein . Pra is autophosphorylated in vitro, with phosphotransfer of the terminal phosphate from {gamma-32P}GTP but not {gamma-32P}ATP . Pra is capable of complex formation with the truncated 12-kDa form of nucleoside diphosphate kinase (Ndk) but not with the 16-kDa form . Purified Pra was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are all present, Pk has a higher affinity than Pra for forming a complex with the 12-kDa Ndk . The 12-kDa Ndk-Pra complex catalyzed increased synthesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk . Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as well when Pra was present in concentrations in excess of that of Pk . Membrane fractions from cells harvested in the mid-log phase demonstrated very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk . Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra . In contrast, membrane fractions of cells harvested at early to late stationary phase showed predominant GTP synthesis and the presence of increasing amounts of Pk and Pra . It is likely that the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression and the survival of P . aeruginosa by modulating the level of GTP. J Mol Biol, 1997 Mar 28, 267(2), 382 - 402 Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design; Campbell AP et al.; The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin . The NMR solution structures of the trans forms of the peptides show conserved beta-turns which have been implicated in antibody and receptor recognition . The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional (1)H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody . Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets . Conformational changes in the beta-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides . Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding . This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P . aeruginosa strains . The binding of the PAK peptide to a disaccharide receptor analog, (beta GalNAc(1-4)beta Gal), was also studied using (1)H NMR in order to map the "adhesintope" recognized by the receptor . Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor . The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host's defence systems. FEBS Lett, 1997 Mar 24, 405(2), 200 - 8 Cystic fibrosis, lung infections, and a human tracheal antimicrobial peptide (hTAP); Ko YH et al.; In order to understand how lungs of healthy people, unlike those of cystic fibrosis (CF) patients, are protected against bacterial infections such as Pseudomonas aeruginosa, the following three key findings were made . First, P . aeruginosa do not multiply when planted onto tracheal epithelial cells from healthy humans but do so profusely on cells from deltaF508 CF patients . Second, some bacteria bind, and gain entrance into CF cells, even at a physiological salt concentration (104 mM) . Third, human tracheal epithelial cells express an approximately 4 kDa peptide (hTAP), which is known in its bovine form to exhibit bactericidal action against P . aeruginosa . A model is proposed depicting both how normal epithelial cells, in a first-line self defense mechanism, may be protected against bacterial infection and how this mechanism may fail during the initial stages of CF. FEMS Microbiol Lett, 1997 Mar 15, 148(2), 217 - 21 Glucose stimulates alginate production and algD transcription in Pseudomonas aeruginosa; Ma JF et al.; A previous study {DeVault et al . (1991) Mol . Microbiol . 5, 2503-2509} suggested that growth of Pseudomonas aeruginosa in glucose-containing medium represses algD gene transcription . In this study, growth of P . aeruginosa in rich medium containing glucose or gluconate increased alginate production and algD transcription at concentrations ranging from 1 to 5%. Biochem Biophys Res Commun, 1997 Mar 6, 232(1), 240 - 6 ATR-FTIR spectroscopic investigation of imipenem-susceptible and -resistant Pseudomonas aeruginosa isogenic strains; Sockalingum GD et al.; The primary mechanism of imipenem resistance in Pseudomonas aeruginosa has been ascribed to an outer membrane impermeability owing to a loss of expression of protein D2 . Attenuated total reflection-Fourier transform infrared spectroscopy in conjunction with statistical methods has been used as a new approach to rapidly discriminate four isogenic strains of P . aeruginosa--susceptible, less susceptible, and highly resistant to imipenem-- and to follow the structural modifications related to this low permeability . Decomposition of the broad protein and carbohydrate contours into underlying Gaussians and comparison of the susceptible and highly resistant strain provided quantitative and ultrastructural information on these strains . This methodology allows for discrimination not of the mutation itself but of its consequences observed in the protein and carbohydrate absorption regions . Its association with other existing biochemical methods may be envisaged since it may allow for rapid orientation of investigations in the field of bacterial resistance diagnosis. Ann Ital Chir, 1997 Mar-Apr, 68(2), 219 - 24 {"Killing" of pseudomonas aeruginosa isolated from patients with critical post-operative complications . Effect of various antibiotics}; Delogu G et al.; We undertook this study to estimate phagocytic killing by neutrophils (PMNs) of Pseudomonas aeruginosa pre-exposed to sub-inhibitory concentration of Amikacin and Imipenem . In particular, we have isolated bacteria from endotracheal aspirates of post-operative patients mechanically ventilated admitted to an ICU with respiratory failure . PMNs were obtained both from these patients (Group A, n . 6) as well as from subjects submitted to surgery with uncomplicated post-operative period (Group B, n . 8) . From specimens tested, 6 strains of Pseudomonas aeruginosa were isolated . Results showed that the rate of killing of bacteria treated with Amikacin was no different from that of untreated bacteria, whichever the source of PMNs, either from Group A or Group B patients . On the other hand, the microbicidal effect on P . aeruginosa exposed to Imipenem was significantly enhanced when PMNs were obtained from Group B patients . In the mixture bacteria, Imipenem and PMNs obtained from Group A the rate of killing was low, similar to the controls without antibiotics . Such a finding suggests a possible impairment of PMNs due to the critical disease and in some way responsible for the host adverse interaction between granulocytes, antibiotics and pathogens . The underlying mechanisms remain to be clarified and further studies are required to understand the possible clinical implications. Pneumologie, 1997 Mar, 51(3), 270 - 3 {Surfactant administration and laterally independent positive pressure ventilation in acute lung failure and atelectasis after septic abortion . Case report}; Hoheisel G et al.; We report the case of a 35 years old female patient suffering from Staphylococcus aureus induced abortion in the 7th/8th week of gestation . Sepsis with acute respiratory failure (ARDS) developed, which could be treated successfully . Pneumonia, caused by Pseudomonas aeruginosa, induced a recurrence of ARDS, complicated by a persistent incomplete atelectasis of the left lung . Independent ventilation of both lungs with increased pressure on the left side combined with bronchoscopy guided instillation of 1 g of bovine surfactant (Alveofact), caused improvement of arterial oxygenation and radiological signs, signalling airation of collapsed lung areas. Jpn J Ophthalmol, 1997 Mar-Apr, 41(2), 63 - 6 Detection of interleukin-1 beta in the tear fluid of patients with corneal disease with or without conjunctival involvement; Fukuda M et al.; To investigate the role of Interleukin-1 (IL-1) in the pathobiology of the cornea, we measured IL-1 beta concentration in tear fluid samples from patients with corneal disease . Twenty patients with unilateral corneal disease were included in the study . Tear fluid samples were collected during the active stages of the disease and following resolution . The fellow (unaffected) eyes served as controls . The concentration of IL-1 beta in the tear fluid samples was measured using a sandwich ELISA method . IL-1 beta was detected in tear fluid from five eyes (three eyes with chemical burns, one with a Pseudomonas aeruginosa corneal ulcer, and one with a peripheral corneal ulcer) at concentrations between 29 and 218 pg/mL . IL-1 beta was not detected in tear fluid from the remaining 15 affected eyes, nor from the control eyes . The detection of IL-1 beta in tear fluid correlated with limbal conjunctival involvement, but did not correlate with the type of disease, size of epithelial defect, or degree of stromal involvement . IL-1 beta in the tear fluid may be one of the factors modifying the complex inflammatory process of the anterior ocular surface. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi, 1997 Mar-Apr, 38(2), 159 - 61 Pseudomonas aeruginosa endophthalmitis in prematurity: report of two cases; Mu SC et al.; Invasive bacterial eye infections in the neonate range from perforating keratitis to endophthalmitis . Endophthalmitis secondary to Pseudomonas aeruginosa has gained clinical and therapeutic importance since mortality rates are high and prognosis concerning preservation of vision is poor, especially in premature infants . We presented two cases with meningitis, septicemia and P . aeruginosa endophthalmitis . If premature infants develop a sepsis-like picture with cloudy cornea and purulent conjunctivitis, we have to consider the possibility of endophthalmitis and do a full ophthalmologic evaluation . Treatment should be started early and consists of systemic antibiotic therapy, as in septicemia . As P . aeruginosa spreads easily, prompt isolation and strict handwashing are indicated. Int J Artif Organs, 1997 Mar, 20(3), 144 - 52 Permeability and adsorption capacity of dialysis membranes to lipid A; Weber C et al.; Hemodialysis membranes were tested in vitro for possible penetration by low molecular weight endotoxins containing lipid A . Using lipid A from Escherichia coli as a model substance for this kind of pyrogen, different dialyzers (F4, E3 . Acepal 1300, Altraflux, F 40, Polyflux 110, Filtral 12, F 60) were challenged by tangential filtration in aqueous medium . All membranes exhibited impermability to lipid A (as well as to LPS from Pseudomonas aeruginosa), which was proved by additional experiments using culture filtrates of Pseudomonas aeruginosa in bicarbonate dialysis fluid, as well as by employing miniaturized dialyzers with synthetic lipid A as a contaminant . Furthermore, the highest adsorption capacities were found for polysulfone and polyamide membranes. Kansenshogaku Zasshi, 1997 Mar, 71(3), 214 - 21 A study of bronchus-associated lymphoid tissue in a rat model of chronic pulmonary infection with Pseudomonas aeruginosa; Kitazawa H et al.; The immunologic pathogenesis of conditions characterized by chronic pulmonary infections, such as diffuse panbronchiolitis and those associated with cystic fibrosis, had not been fully clarified . Organized lymphoid tissue along the airway has been termed bronchus-associated lymphoid tissue (BALT), and hyperplasia of BALT is frequently observed in chronic pulmonary infections in humans . To investigate the role of BALT, we intratracheally inoculated rats with Pseudomonas aeruginosa (PA) enmeshed in agar beads according to the method of Cash et al., thereby establishing a chronic pulmonary infection model . Histopathological examination of tissue from this rat model revealed the accumulation of lymphocytes and foamy cells around bronchioles . This finding corresponds to chronic bronchiolitis in humans . Hyperplasia of BALT was also observed . Immunohistochemical examination demonstrated that Ia+ cells, helper T cells, surface IgM-positive (sIgM+) cells and sIgA+ cells had gradually increased in BALT and the walls of peripheral airways during the period from day 4 to 7 . The anti-PA IgA antibody titer in bronchoalveolar lavage fluid (BALF) was also elevated during this period . After day 21, non-helper T cells became predominant in tissue sections, and the numbers of various immunoglobulin-positive cells as well as the anti-PA IgA antibody titer in BALF were reduced . Histological examination revealed that the inflammatory findings had also diminished . The time course of changes in the various immune cells in BALT and the walls of peripheral airways, paralleled the reductions in anti-PA IgA antibody titers in BALF . Our findings suggest that hyperplastic BALT may be one source of the Ig producing cells which play an important role in the local immune response characteristic of chronic pulmonary infections. Am J Respir Crit Care Med, 1997 Mar, 155(3), 928 - 36 The effects of post-treatment with lisofylline, a phosphatidic acid generation inhibitor, on sepsis-induced acute lung injury in pigs; Hasegawa N et al.; The effects of lisofylline {(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine} (LSF), an inhibitor of de novo phosphatidic acid (PA) generation, on sepsis-induced acute lung injury was studied using Hanford minipigs weighing 18 to 25 kg . Sepsis was induced by an intravenous infusion of Pseudomonas aeruginosa (1 x 10(6)/colony-forming units/kg/min over 2 h) . Saline was used as the control vehicle . Six groups were studied: saline control group (SALINE: n = 5); sepsis control group (SEPSIS: n = 5); LSF control group (LSF: n = 5), which received a 25-mg/kgbolus of LSF 30 min before time zero followed by continuous infusion of 10 mg/kg/h throughout the study; LSF-treated septic groups, which were treated with LSF 30 min prior to sepsis (Pre: n = 5), 1 h postonset (Post-1 h: n = 8) or h postonset (Post-2 h: n = 8) of the bacterial infusion . Hemodynamics PaO2, neutrophil counts, and plasma porcine tumor necrosis factor-alpha concentrations were monitored for 6 h . After the minipigs were killed, lung tissue was sampled to measured wet-to-dry weight ratio (W/D), tissue albumin index (TAI), thiobarbituric acid-reactive material content (TBARM), and myeloperoxidase (MPO) activity . Compared with the SALINE group, the SEPSIS group showed significant systemic hypotension, pulmonary hypertension, arterial hypoxemia, neutropenia, and increase in TNF-alpha, MPO activity, W/D, TBARM, and TAI . LSF treatment attenuated sepsis-induced pulmonary hypertension, neutropenia, and hypoxemia, and increased MPO activity and lung injury measurements in the Pre and Post-1 h groups, but its efficacy was blunted in the Post-2 h group . Plasma TNF-alpha was decreased only in the Pre group . Thus, inhibition of intracellular PA generation through de novo pathways attenuates sepsis-induced acute lung injury. Biomaterials, 1997 Mar, 18(6), 503 - 10 Role of physiological conditions in the oropharynx on the adherence of respiratory bacterial isolates to endotracheal tube poly(vinyl chloride); Jones DS et al.; Pneumonia is a major problem in intensive care patients and can be induced by pathogenic bacteria adhering to poly(vinyl chloride) (PVC) endotracheal (ET) tubes . This study examines the influence of surface properties on the adherence of the respiratory isolates Staphylococcus aureus and Pseudomonas aeruginosa to PVC . In particular, the influence of respiratory tract physiological conditions, 5% CO2 and saliva, on adherence was investigated . In general, decreased adherence to PVC was observed when bacteria were grown in CO2 . When these CO2-grown bacteria were treated with saliva their adherence to PVC significantly increased; however, their adherence was significantly reduced to saliva-treated PVC . Treatment of both bacterial isolates with saliva decreased their negative zeta potential, a factor which may directly contribute to the observed increased microbial (saliva pretreated) adherence to PVC . Cell surface hydrophobicity (CSH) was evaluated by measuring the initial rates of microbial removal from a buffered aqueous phase, to ensure the absence of electrostatic interactions, to an organic phase (xylene) . Under physiological conditions, CSH did not appear to be a dominant factor in biomaterial adherence as the CSH of S . aureus was decreased by saliva treatment but was unchanged for Ps . aeruginosa . Additionally, CSH also differed for the two isolates when grown in CO2, significantly decreasing with S . aureus but remaining unaltered with Ps . aeruginosa . Saliva treatment of PVC also decreased the advancing and receding contact angles of the biomaterial and its surface roughness, which may be a factor in the decreased adherence of saliva-treated bacteria to this surface . Alternative biomaterials or surface modifications appear necessary for the desired improvements in ET tube effectiveness . This study highlights the influence of physiological conditions on biomaterial and bacterial surface characteristics and subsequent interactions . It is imperative that the physiological conditions predominating in the clinical area of biomaterial use be considered when investigating device biocompatibility. Pharmazie, 1997 Mar, 52(3), 238 - 9 Postantibiotic effect of norfloxacin and its influence on profiles of outer membrane proteins of Pseudomonas aeruginosa; Hostacka A et al.; Norfloxacin at suprainhibitory concentrations induced postantibiotic effects (PAEs) in the range of 6 h to 11.4 h (2.MIC) and of 10.1 to more than 13.4 h (4, MIC) against three P . aeruginosa strains . After PAEs, the outer membrane profile proteins of the strains studied was changed . Overproduction of 41 kDa protein as well as strong reduction of 23 kDa and 45 kDa proteins were found in all three strains . A reduction of the protein band in the range of 35 kDa was also observed in all strains, but to a smaller extent. Microbiol Mol Biol Rev, 1997 Mar, 61(1), 47 - 64 Microbial production of surfactants and their commercial potential; Desai JD et al.; Many microorganisms, especially bacteria, produce biosurfactants when grown on water-immiscible substrates . Biosurfactants are more effective, selective, environmentally friendly, and stable than many synthetic surfactants . Most common biosurfactants are glycolipids in which carbohydrates are attached to a long-chain aliphatic acid, while others, like lipopeptides, lipoproteins, and heteropolysaccharides, are more complex . Rapid and reliable methods for screening and selection of biosurfactant-producing microorganisms and evaluation of their activity have been developed . Genes involved in rhamnolipid synthesis (rhlAB) and regulation (rhlI and rhlR) in Pseudomonas aeruginosa are characterized, and expression of rhlAB in heterologous hosts is discussed . Genes for surfactin production (sfp, srfA, and comA) in Bacillus spp . are also characterized . Fermentative production of biosurfactants depends primarily on the microbial strain, source of carbon and nitrogen, pH, temperature, and concentration of oxygen and metal ions . Addition of water-immiscible substrates to media and nitrogen and iron limitations in the media result in an overproduction of some biosurfactants . Other important advances are the use of water-soluble substrates and agroindustrial wastes for production, development of continuous recovery processes, and production through biotransformation . Commercialization of biosurfactants in the cosmetic, food, health care, pulp- and paper-processing, coal, ceramic, and metal industries has been proposed . However, the most promising applications are cleaning of oil-contaminated tankers, oil spill management, transportation of heavy crude oil, enhanced oil recovery, recovery of crude oil from sludge, and bioremediation of sites contaminated with hydrocarbons, heavy metals, and other pollutants . Perspectives for future research and applications are also discussed. Arzneimittelforschung, 1997 Mar, 47(3), 307 - 10 Synthesis and antimicrobial evaluation of indole containing derivatives of 1,3,4-thiadiazole, 1,2,4-triazole and their open-chain counterparts; Tsotinis A et al.; The increasing clinical importance of drug-resistant bacterial pathogens has lent additional urgency to microbiological and antibacterial research . New indolic derivatives of triazoles, thiadiazoles and their respective open-chain thiosemicarbazides were evaluated for antibacterial and antifungal activity . The microorganisms used were the Gram-negative bacteria Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853, the Gram-positive bacteria Staphylococcus aureus ATCC 25923 and Bacillus subtilis BBL 12084 and the yeasts Candida and Saccharomyces cerevisiae ATCC 2366 . The most potent compounds were indole derivatives (12a-c) bearing 1,2,4-triazo-thien-5-yl moiety, which exhibit interesting antibacterial and antifungal activities. Vet Microbiol, 1997 Mar, 54(3-4), 275 - 85 The occurrence of Pseudomonas aeruginosa in fleece washings from sheep affected and unaffected with fleece rot; Kingsford NM et al.; Fleece rot is an exudative bacterial dermatitis in sheep associated with and initiated by prolonged wetting of the skin . It is the major predisposing disease for flystrike (cutaneous myasis) . Based on limited data in the literature, P . aeruginosa is considered to be the main causative organism of fleece rot . This study investigated the occurrence of P . aeruginosa in fleece washings from sheep affected and unaffected with fleece rot under field conditions . Three field surveys of a total of 1568 sheep showed that 646 (41%) were affected with fleece rot and of these 646 sheep . P . aeruginosa could not be detected in 554 (86%) . This suggests that other fleece bacteria play a significant role in fleece rot . However, the surveys consistently showed that the presence of P . aeruginosa was associated with increased severity of fleece rot and subsequent flystrike . Although it might only contribute in part to the disease complex, the importance of P . aeruginosa can not be underestimated and warrants consideration for inclusion in future fleece rot vaccines. Vet Microbiol, 1997 Mar, 54(3-4), 255 - 74 Antigens for serological diagnosis of ovine footrot; Whittington RJ et al.; An antigen extracted from Dichelobacter nodosus with potassium thiocyanate (KSCN) is currently used in enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of ovine footrot, but the test lacks specificity in mature sheep . Other antigens were therefore evaluated for use in this test . Structural components of the cell envelope of D . nodosus including outer membrane, cytoplasmic membrane, lipopolysaccharide and pilus and extracellular proteases were purified from cultured D . nodosus while recombinant membrane proteins, protease and pilus antigens were also evaluated . Many antigenic components of D . nodosus participated in reactions in ELISA that were not specific for infection with D . nodosus and apart from pilus, none of the antigens resulted in improved specificity of the ELISA . Using a positive-negative cut-off to yield sensitivity of 70%, ELISA using pili from cultured D . nodosus serogroup A had a specificity of 98.3% compared with 89.7% for the ELISA with KSCN-extract as antigen (P < 0.001) . Recombinant pili morphogenetically expressed in Pseudomonas aeruginosa were unsuitable for use in ELISA due to copurification of Pseudomonas antigens to which apparently healthy sheep directed antibodies . The application of ELISA with D . nodosus pilus as antigen in footrot control programs is discussed. J Antimicrob Chemother, 1997 Mar, 39(3), 325 - 30 Susceptibility of Pseudomonas aeruginosa of various pyocin types to the newly synthesized ampicillin derivative, N-(6,7-difluoroquinolonyl)ampicillin; Chen CH et al.; Six hundred and thirty-two isolates of Pseudomonas aeruginosa of 17 pyocin types were collected in 1993 in Taiwan . Types 1, 10, 3, 35 and 12 were the most common pyocin types identified in Taiwan with isolation frequencies of 47.3%, 24.4%, 7.6%, 3.6% and 2.2%, respectively . Several pyocin subtypes were determined . All pyocin types (one isolate of each tested) were resistant to ampicillin and nalidixic acid, but sensitive to fluoroquinolone antibiotics, such as norfloxacin and enoxacin, indicating that cross-resistance to quinolone antibiotics of nalidixic acid and fluoroquinolone derivatives has not developed . A new ampicillin derivative of 6,7-difluoroquinolonic acid, N-(6,7-difluoroquinolonyl)-ampicillin (AU-1), was synthesized by coupling ampicillin with 6,7-difluoroquinolonic acid (FP-3) . Compound AU-1 was much more active than either ampicillin or FP-3 alone against all pyocin types of P . aeruginosa and induced filamentation in most growing cells. J Antimicrob Chemother, 1997 Mar, 39(3), 309 - 17 Sensitivity testing of ciprofloxacin for Pseudomonas aeruginosa; Ibrahim-Elmagboul IB et al.; UK clinical laboratories overestimate ciprofloxacin resistance amongst Pseudomonas aeruginosa isolates, relative to the MIC breakpoint of 1 mg/L . Most tests leading to this overestimation use 1 microg discs and are by Stokes' method with the breakpoint taken as the zone radius for P . aeruginosa NCTC 10662 minus 3 mm . Aiming to reduce this error rate, we examined alternative disc breakpoints . Tests were performed for 100 P . aeruginosa isolates on three media, with breakpoints selected (i) as the zone for P . aeruginosa NCTC 10662 minus 7 mm, as recommended for ciprofloxacin by the BSAC; (ii) with reference to MIC/zone correlation lines; (iii) from natural divisions in zone distribution histograms; and (iv) so as to minimize categorization errors . Breakpoints from regression lines, and those optimized to the susceptibility distribution, reduced the proportion of susceptible organisms misreported as resistant, but the improvement was not significant (P > 0.05, chi2 test) . The breakpoint of the zone radius for P . aeruginosa NCTC 10662 minus 7 mm significantly reduced (P < 0.05) the number of susceptible organisms reported as resistant, but led to 50-75% of those with low level resistance (MIC 2-4 mg/L) and 4-10% of those with high-level resistance (MIC > 4 mg/L) being classed as susceptible . Irrespective of the medium and the basis of choosing breakpoints, 5 microg ciprofloxacin discs gave a lower rate of susceptible organisms being reported as resistant than did 1 microg discs; however, the improvement was not significant (P > 0.05, chi2 test) and the 5 microg discs had the disadvantages of forming very large zones for susceptible isolates and giving some--albeit small--zones for highly resistant organisms . In conclusion, the over-reporting of resistance could be reduced by use of zone breakpoints optimized to the MIC distribution and by the use of 5 microg discs, but the case for these changes is not overwhelming; taking the breakpoint as the zone for NCTC 10662 minus 7 mm led to unacceptable numbers of resistant organisms being reported as susceptible . More fundamentally, ciprofloxacin zones and MICs are continuously distributed for P . aeruginosa isolates, so susceptibility tests cannot divide the species into discrete populations . In these circumstances, it is optimistic to expect disc and MIC categorizations to agree perfectly. Proteins, 1997 Mar, 27(3), 385 - 94 The metal site of Pseudomonas aeruginosa azurin, revealed by a crystal structure determination of the Co(II) derivative and Co-EPR spectroscopy; Bonander N et al.; The crystal structure of cobalt-substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 A resolution . There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers . The dimer packing is very similar to that of the wild-type Pseudomonas aeruginosa azurin dimer . Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood . Some differences are obvious, however . In wild-type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,S(delta) atom is increased to 3.3-3.5 A and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1-2.4 A . The X-band EPR spectrum of the high-spin Co(II) in azurin is well resolved (apparent g values gx' = 5.23; gy' = 3.83; gz' = 1.995, and hyperfine splittings Ax' = 31; Ay' = 20-30; Az' = 53 G) and indicates that the ligand field is close to axial. Thorax, 1997 Mar, 52(3), 260 - 4 Effects of airway infection by Pseudomonas aeruginosa: a computed tomographic study; Miszkiel KA et al.; BACKGROUND: Pseudomonas aeruginosa commonly infects the airways of patients with bronchiectasis . A study was undertaken to examine the relationship between infection of the airways with this pathogen, the morphological pattern of bronchiectasis on thin section computed tomographic (CT) scanning, symptom duration, smoking habits of the patients, and the presence of airflow obstruction . METHODS: Thin section CT scans of 22 adult patients with bronchiectasis and concurrent sputum infected by P aeruginosa (Pa +ve) and those of 45 randomly selected patients not infected by P aeruginosa (Pa -ve) were analysed independently by two thoracic radiologists . Patients with cystic fibrosis were excluded . Each scan was scored at a lobar level for extent of bronchiectasis, severity of bronchial wall thickening and dilatation, predominant pattern of bronchiectasis, presence of mucus plugging, and degree of decreased attenuation of the lung parenchyma . RESULTS: The Pa +ve group had more extensive bronchiectasis and a greater degree of bronchial wall thickening and dilatation on the CT scan than the Pa -ve group; more extensive decreased attenuation was seen in the Pa +ve group . These findings were robust on multivariate analysis; decreased attenuation was also independently related to the duration of sputum production . CONCLUSION: Patients with bronchiectasis infected by P aeruginosa have more extensive and severe bronchiectasis on thin section CT scanning than those without P aeruginosa infection . The bronchi and small airways are both involved, reflecting the end result of complex interactions between host airways and the numerous virulence factors produced by P aeruginosa. Can J Microbiol, 1997 Mar, 43(3), 220 - 6 Overexpression, purification, and analysis of the c1 repressor protein of Pseudomonas aeruginosa bacteriophage D3; Farinha MA et al.; A 3.1-kb region of the bacteriophage D3 genome which contains the immunity functions has recently been sequenced (GenBank accession No . L22692) . Sequence analysis indicated the presence of a putative repressor gene (c1) whose protein product functions to maintain the bacteriophage genome as a stably integrated prophage in the chromosome of Pseudomonas aeruginosa . A plasmid was constructed that overexpresses repressor C1 protein under control of P(tac) in Escherichia coli . C1 protein was subsequently purified and characterized as a 223 amino acid protein with specific binding affinity for 14-base imperfect palindromic operator sequences located on the genome of bacteriophage D3 . N-terminal protein sequence data obtained from automated Edman degradation (16 cycles) of purified repressor protein were identical to the predicted sequence based on DNA sequence analysis of the c1 open reading frame. Chemotherapy, 1997 Mar-Apr, 43(2), 118 - 22 Outer membrane proteins and elastase of Pseudomonas aeruginosa after the postantibiotic effect induced by amikacin; Hostacka A et al.; Postantibiotic effects were induced in three Pseudomonas aeruginosa strains after short-term treatment (30 min) with amikacin at suprainhibitory concentrations in the range of 0.6-1.2 h (2 x MIC) and of 3.0-4.4 h (4 x MIC) . Significant suppression of P . aeruginosa elastase activity was found after treatment with amikacin at 4 x MIC concentration (to 10-69.2% of the control values); 2 x MIC was less effective . The profile of outer membrane proteins of P . aeruginosa was not changed after treatment with amikacin at both concentrations. Eur J Pediatr, 1997 Mar, 156(3), 209 - 11 Silastic catheters for home antibiotic therapy in patients with cystic fibrosis; Buck C et al.; Repeated 14-day courses of intravenous antibiotic therapy for patients with cystic fibrosis (CF), who have been colonized with Pseudomonas aeruginosa (PA), is one currently accepted treatment . Conventional intravenous cannulas for antibiotic delivery often have a short line life leading to frequent venipunctures . Therefore we used silastic catheters as a peripheral venous access . Silastic catheters (15 cm, 0.6 mm diameter) were inserted 10 cm into a cubital vein in 15 patients with CF (age 5-32 years) for 20 antibiotic courses . After the antibiotic infusion the catheter was flushed with 200 U heparin (2 ml Vetren) . In all patients the antibiotic therapy was delivered as a home therapy . In 15 antibiotic courses the silastic catheter could be continuously used for 14 days . One patient with methicillin resistant Staphylococcus aureus received antibiotic therapy for 54 consecutive days using the same silastic catheter . The catheter had to be removed in four courses: once because of thrombophlebitis with local inflammation, once because of burning pain during infusion and occlusion twice . In one case the patient removed his catheter because of technical problems . No other serious side effects occurred . Ten patients had previously received intravenous antibiotics at least once . The median line life of the last used conventional peripheral cannula of all patients was 4 days versus 14 days with the use of the silastic catheter (P < 0.005) . All patients preferred the silastic catheter to other venous access . CONCLUSION: Because of the long line life and easy handling, silastic catheters may be an alternative venous access to perform home antibiotic therapy in patients with CF. Lett Appl Microbiol, 1997 Mar, 24(3), 169 - 71 Multiple antibiotic resistance (MAR) index and its reversion in Pseudomonas aeruginosa; Paul S et al.; The MAR indexes of hospital isolates of Pseudomonas aeruginosa were determined with reference to nine different cephalosporins . The values for all the strains were higher than 0.2 suggesting their origin from a high risk source of contamination where antibiotics are often used . Emergence of MAR pathogenic strains of Ps . aeruginosa indicated possible nosocomial infection in the hospital environment . beta-Lactamases produced by these organisms were tested and their inhibition by clavulanic acid was studied . beta-Lactamase produced by one of these strains (Ps-1) could not be inhibited by clavulanic acid whereas beta-lactamases of three other strains (Ps-2, Ps-3 and Ps-4) could be inhibited by clavulanic acid in the presence of cephalosporins, suggesting a possible use of clavulanic acid in combination with cephalosporins, to combat beta-lactamase induced resistance in Ps . aeruginosa. J Bacteriol, 1997 Mar, 179(6), 2029 - 37 Cloning, expression, and purification of UDP-3-O-acyl-GlcNAc deacetylase from Pseudomonas aeruginosa: a metalloamidase of the lipid A biosynthesis pathway; Hyland SA et al.; The lpxC (envA) gene of Escherichia coli encodes UDP-3-O-acyl-GlcNAc deacetylase, the second and committed step of lipopolysaccharide biosynthesis . Although present in all gram-negative bacteria examined, the deacetylase from E . coli is the only example of this enzyme that has been expressed and purified . In order to examine other variants of this protein, we cloned the Pseudomonas aeruginosa deacetylase structural gene from a lambda library as a 5.1-kb EcoRI fragment . The LpxC reading frame encodes an inferred protein of 33,435 Da that is highly homologous to the E . coli protein and that possesses a nearly identical hydropathy profile . In order to verify function, we subcloned the P . aeruginosa lpxC gene into the T7-based expression vector pET11a . Upon induction at 30 degrees C, this construct yielded active protein to approximately 18% of the soluble fraction . We devised a novel, rapid, and reproducible assay for the deacetylase which facilitated purification of the enzyme in three steps . The purified recombinant protein was found to be highly sensitive to EDTA yet was reactivated by the addition of excess heavy metal, as was the case for crude extracts of P . aeruginosa . In contrast, deacetylase activity in crude extracts of E . coli was insensitive to EDTA, and the extracts of the envA1 mutant were sensitive in a time-dependent manner . The lpxC gene has no significant homology with amidase signature sequences . Therefore, we assign this protein to the metalloamidase family as a member with a novel structure. Clin Diagn Lab Immunol, 1997 Mar, 4(2), 147 - 55 Phage display and bacterial expression of a recombinant Fab specific for Pseudomonas aeruginosa serotype O6 lipopolysaccharide; Tout NL et al.; Immunotherapy with antibodies (Abs) against the lipopolysaccharide (LPS) of Pseudomonas aeruginosa remains an alternative to serotype-specific LPS-based vaccines due to their limited use and to antibiotics due to the intrinsic resistance to antimicrobials observed in P . aeruginosa . We have chosen a monoclonal Ab (MAb), MF23-1, that binds to the O antigen of the most clinically relevant serotype, IATS O6, for producing a recombinant antibody . Heavy (H) and light (L) chain genes were isolated from MF23-1 to form a functional Fab molecule in the periplasm of Escherichia coli and on the surface of phage by using phagemid vector pComb3 . The entire kappa L chain gene was used, but the H chain gene was amplified to 2 amino acids past cysteine 128 which is involved in interchain disulfide bond formation with the L chain . The truncated H chain associated with the L chain in the periplasm of E . coli to form a functional Fab molecule that bound in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay to O6 LPS . Therefore, the remainder of the CH1 past cysteine 128 is not essential for stable formation of the Fab portion of MF23-1 . This recombinant Fab (r-Fab) was shown to be specific for the LPS of the most predominant clinical isolate, serotype O6, while no cross-reactivity was detected to the LPS of the other 19 remaining serotypes . This r-Fab was also expressed on the surface of filamentous phage upon addition of helper phage to recombinant E . coli containing phagemid . Recombinant phage from clones MT13 and MT24 bound specifically to O6 LPS in ELISA . These results represent an important step toward the design of therapeutic Abs to be used against P . aeruginosa infections. Biochem J, 1997 Mar 1, 322 ( Pt 2), 625 - 31 Expression, purification and kinetic characterization of wild-type human ornithine transcarbamylase and a recurrent mutant that produces 'late onset' hyperammonaemia; Morizono H et al.; Ornithine Transcarbamylase Deficiency, an X-linked disorder, is the most common cause of inherited urea cycle disorders . Approx . 90 mutations that produce reduced levels of ornithine transcarbamylase (OTCase) activity have been identified in patients {Tuchman (1993) Hum . Mutat . 2, 174-178; Tuchman and Plante (1995) Hum . Mutat . 5, 293-295} . A model of the three-dimensional structure of OTCase, developed on the basis of its homology to the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase) {Tuchman, Morizono, Reish, Yuan and Allewell (1995) J . Med . Genet . 32, 680-688}, and in good agreement with the crystal structure of Pseudomonas aeruginosa OTCase {Villeret, Tricot, Stalon and Dideberg (1995) Proc . Natl . Acad . Sci . U.S.A . 92, 10762-10766}, indicates that many mutations that produce severe clinical symptoms are at the active site or buried in the interior of the protein . However, one of the few recurrent mutations, R277W, an alteration that produces a milder phenotype of ornithine transcarbamylase deficiency, is located in the model in a loop remote from the active site that is analogous to a similar loop (the 240's loop, a flexible loop of the catalytic chain of Escherichia coli aspartate transcarbamylase, comprised of residues 230-250) of ATCase . Human wild-type OTCase and the R277W mutant have been cloned and overexpressed in E . coli and a rapid and efficient purification method utilizing the bisubstrate analogue, Ndelta-(phosphonacetyl)-L-ornithine, has been developed and used to purify both proteins . Gel chromatography indicates both are trimeric . The pH dependence of the kinetic parameters of the wild-type enzyme is similar to that of E . coli OTCase {Kuo, Herzberg and Lipscomb (1985) Biochemistry 24, 4754-4761}, suggesting that its catalytic mechanism is similar, although its maximal activity is approx . 10-fold less . Compared with the wild-type, the R277W mutant has nearly 70-fold lower affinity for L-ornithine, shows no substrate inhibition, and its thermal stability is reduced by 5 degrees C . Its reduced affinity for L-ornithine, which in turn results in lower activity at physiological concentrations of ornithine, as well as its reduced stability, may contribute to the clinical effects that it produces. J Bacteriol, 1997 Mar, 179(5), 1646 - 54 Functional analysis of exsC and exsB in regulation of exoenzyme S production by Pseudomonas aeruginosa; Goranson J et al.; Expression of ExsC, ExsB, and ExsA (the exoenzyme S trans-regulatory locus) of Pseudomonas aeruginosa was analyzed by using complementation, RNase protection, translational fusion, and T7-directed protein expression analyses . T7 expression analyses in E . coli hosts demonstrated that ExsC, ExsA, and a truncated form of ExsD (a partial open reading frame located 3' of ExsA) were translated; however, a product corresponding to ExsB was undetectable . T7-mediated transcription and translation of the antisense strand resulted in production of a 18.5-kDa product, termed ExsB', which overlapped the predicted ExsB product . In complementation experiments, deletion of the region encoding ExsB and most of ExsB' severely reduced exoenzyme S production . Site-specific mutagenesis of the start codons for ExsB and ExsB', however, did not affect exoenzyme S production . RNase protection studies were initiated to examine the hypothesis that RNA encoded within the ExsB/ExsB' region exerted a regulatory effect . RNA encoding ExsB' was not detectable from chromosomal genes or complementation constructs, indicating that ExsB' was not expressed in P . aeruginosa . To determine the pattern of translation, a chloramphenicol acetyltransferase gene (cat) reporter was fused in frame with ExsB and with ExsA in the context of the entire locus or in the absence of the exsB region . These experiments indicated that exsB was not translated but that deletion of the exsB region affected the translation of ExsA-CAT . RNase protection assays further suggested that deletion of exsB resulted in a processing of ExsA mRNA . Our data indicate that the untranslated exsB region of the trans-regulatory locus mRNA mediates either the stability or the translation of exsA . Complementation analysis further suggests that ExsC may play a role in the translation or stability of ExoS. J Bacteriol, 1997 Mar, 179(5), 1609 - 13 Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa; Liu S et al.; Genetic studies have shown that the 53-kDa (Exo53) and 49-kDa (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa are encoded by separate genes, termed exoT and exoS, respectively . Although ExoS and Exo53 possess 76% primary amino acid homology, Exo53 has been shown to express ADP-ribosyltransferase activity at about 0.2% of the specific activity of ExoS . The mechanism for the lower ADP-ribosyltransferase activity of Exo53 relative to ExoS was analyzed by using a recombinant deletion protein which contained the catalytic domain of Exo53, comprising its 223 carboxyl-terminal residues (termed N223-53) . N223-53 was expressed in Escherichia coli as a stable, soluble fusion protein which was purified to >80% homogeneity . Under linear velocity conditions, N223-53 catalyzed the FAS (for factor activating exoenzyme S)-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) at 0.4% and of the Ras protein at 1.0% of the rates of catalysis by N222-49 . N222-49 is a protein comprising the 222 carboxyl-terminal residues of ExoS, which represent its catalytic domain . N223-53 possessed binding affinities for NAD and SBTI similar to those of N222-49 (less than fivefold differences in Kms) but showed a lower velocity rate for the ADP-ribosylation of SBTI . This indicated that the primary defect for ADP-ribosylation by Exo53 resided within its catalytic capacity . Analysis of hybrid proteins, composed of reciprocal halves of N223-53 and N222-49, localized the catalytic defect to residues between positions 235 and 349 of N223-53 . E385 was also identified as a potential active site residue of Exo53. J Bacteriol, 1997 Mar, 179(5), 1490 - 6 Identification of a penicillin-binding protein 3 homolog, PBP3x, in Pseudomonas aeruginosa: gene cloning and growth phase-dependent expression; Liao X et al.; A homolog of Pseudomonas aeruginosa penicillin-binding protein 3 (PBP3), named PBP3x in this study, was identified by using degenerate primers based on conserved amino acid motifs in the high-molecular-weight PBPs . Analysis of the translated sequence of the pbpC gene encoding this PBP3x revealed that 41 and 48% of its amino acids were identical to those of Escherichia coli and P . aeruginosa PBP3s, respectively . The downstream sequence of pbpC encoded convergently transcribed homologs of the E . coli soxR gene and the Mycobacterium bovis adh gene . The pbpC gene product was expressed from the T7 promoter in E . coli and was exported to the cytoplasmic membrane of E . coli cells and could bind {3H} penicillin . By using a broad-host-range vector, pUCP27, the pbpC gene was expressed in P . aeruginosa PAO4089 . {3H}penicillin-binding competition assays indicated that the pbpC gene product had lower affinities for several PBP3-targeted beta-lactam antibiotics than P . aeruginosa PBP3 did, and overexpression of the pbpC gene product had no effect on the susceptibility to the PBP3-targeted antibiotics tested . By gene replacement, a PBP3x-defective interposon mutant (strain HC132) was obtained and confirmed by Southern blot analysis . Inactivation of PBP3x caused no changes in the cell morphology or growth rate of exponentially growing cells, suggesting that pbpC was not required for cell viability under normal laboratory growth conditions . However, the upstream sequence of pbpC contained a potential sigma(s) recognition site, and pbpC gene expression appeared to be growth rate regulated . {3H}penicillin-binding assays indicated that PBP3 was mainly produced during exponential growth whereas PBP3x was produced in the stationary phase of growth. J Bacteriol, 1997 Mar, 179(5), 1452 - 9 An operon containing fumC and sodA encoding fumarase C and manganese superoxide dismutase is controlled by the ferric uptake regulator in Pseudomonas aeruginosa: fur mutants produce elevated alginate levels; Hassett DJ et al.; The activities of fumarase- and manganese-cofactored superoxide dismutase (SOD), encoded by the fumC and sodA genes in Pseudomonas aeruginosa, are elevated in mucoid, alginate-producing bacteria and in response to iron deprivation (D . J . Hassett, M . L . Howell, P . A . Sokol, M . L . Vasil, and G . E . Dean, J . Bacteriol . 179:1442-1451, 1997) . In this study, a 393-bp open reading frame, fagA (Fur-associated gene), was identified immediately upstream of fumC, in an operon with orfX and sodA . Two iron boxes or Fur (ferric uptake regulatory protein) binding sites were discovered just upstream of fagA . Purified P . aeruginosa Fur caused a gel mobility shift of a PCR product containing these iron box regions . DNA footprinting analysis revealed a 37-bp region that included the Fur binding sites and was protected by Fur . Primer extension analysis and RNase protection assays revealed that the operon is composed of at least three major iron-regulated transcripts . Four mucoid fur mutants produced 1.7- to 2.6-fold-greater fumarase activity and 1.7- to 2.3-greater amounts of alginate than wild-type organisms . A strain devoid of the alternative sigma factor AlgT(U) produced elevated levels of one major transcript and fumarase C and manganase-cofactored SOD activity, suggesting that AlgT(U) may either play a role in regulating this transcript or function in some facet of iron metabolism . These data suggest that the P . aeruginosa fagA, fumC, orfX, and sodA genes reside together on a small operon that is regulated by Fur and is transcribed in response to iron limitation in mucoid, alginate-producing bacteria. J Bacteriol, 1997 Mar, 179(5), 1442 - 51 Fumarase C activity is elevated in response to iron deprivation and in mucoid, alginate-producing Pseudomonas aeruginosa: cloning and characterization of fumC and purification of native fumC; Hassett DJ et al.; We report the discovery of fumC, encoding a fumarase, upstream of the sodA gene, encoding manganese superoxide dismutase, in Pseudomonas aeruginosa . The fumC open reading frame, which terminates 485 bp upstream of sodA, contains 1,374 bp that encode 458 amino acids . A second 444-bp open reading frame located between fumC and sodA, called orfX, showed no homology with any genes or proteins in database searches . A fumarase activity stain revealed that P . aeruginosa possesses at least two and possibly three fumarases . Total fumarase activity was at least approximately 1.6-fold greater in mucoid, alginate-producing bacteria than in nonmucoid bacteria and decreased 84 to 95% during the first 5 h of aerobic growth, followed by a rapid rise to maximum activity in stationary phase . Bacteria exposed to the iron chelator 2,2'-dipyridyl, but not ferric chloride, demonstrated an increase in fumarase activity . Mucoid bacteria produced approximately twofold-higher levels of the siderophores pyoverdin and pyochelin than nonmucoid bacteria . Northern blot analysis revealed a transcript that included fumC, orfX, and sodA, the amount of which was increased in response to iron deprivation . A P . aeruginosa fumC mutant produced only approximately 40% the alginate of wild-type bacteria . Interestingly, a sodA mutant possessed an alginate-stable phenotype, a trait that is typically unstable in vitro . These data suggest that mucoid bacteria either are in an iron-starved state relative to nonmucoid bacteria or simply require more iron for the process of alginate biosynthesis . In addition, the iron-regulated, tricarboxylic acid cycle enzyme fumarase C is essential for optimal alginate production by P . aeruginosa. Infect Immun, 1997 Mar, 65(3), 1071 - 6 Augmentation of oxidant injury to human pulmonary epithelial cells by the Pseudomonas aeruginosa siderophore pyochelin; Britigan BE et al.; Pseudomonas aeruginosa causes acute and chronic infections of the human lung, with resultant tissue injury . We have previously shown that iron bound to pyochelin, a siderophore secreted by the organism to acquire iron, is an efficient catalyst for hydroxyl radical (HO.) formation and augments injury to pulmonary artery endothelial cells resulting from their exposure to superoxide (O2.) and/or H2O2 . Sources for O2- . and H2O2 included phorbol myristate acetate (PMA)-stimulated neutrophils and pyocyanin . Pyocyanin, another P . aeruginosa secretory product, undergoes cell-mediated redox, thereby forming O2- . and H2O2 . In P . aeruginosa lung infections, damage to airway epithelial cells is probably more extensive than that to endothelial cells . Therefore, we examined whether ferripyochelin also augments oxidant-mediated damage to airway epithelial cells . A549 cells, a human type II alveolar epithelial cell line, was exposed to H2O2, PMA-stimulated neutrophils, or pyocyanin, and injury was determined by release of 51Cr from prelabeled cells . Ferripyochelin significantly increased (> 10-fold) oxidant-mediated cell injury regardless of whether H2O2, neutrophils, or pyocyanin was employed . Apo-pyochelin was not effective, and ferripyochelin was not toxic by itself at the concentrations employed . Spin trapping with alpha-(4-pyrridyl-1-oxide)-N-t-butyl-nitrone-ethanol confirmed the generation of HO., and injury was decreased by a variety of antioxidants, including superoxide dismutase, catalase, and dimethylthiourea . These data are consistent with the hypothesis that the presence of ferripyochelin at sites of P . aeruginosa lung infection could contribute to tissue injury through its ability to promote HO.-mediated damage to airway epithelial cells. J Mol Biol, 1997 Feb 21, 266(2), 357 - 66 Crystal structures of modified apo-His117Gly and apo-His46Gly mutants of Pseudomonas aeruginosa azurin; Hammann C et al.; The X-ray crystal structures of two metal ligand mutants of azurin from Pseudomonas aeruginosa have been solved . In both mutants (His117Gly and His46Gly azurin) one of the copper coordinating histidine residues is replaced by a glycine, creating an empty space in the coordination sphere of the copper ion . The crystal structure of His117Gly azurin at 2.4 A resolution showed that this mutant had undergone partial oxidation at the disulfide bridge between Cys3 and Cys26 and full oxidation at the copper ligand Cys112 . There is no copper present in the crystallized form and the bulky group of the oxidized cysteine at position 112 causes large structural rearrangements in the protein structure, especially in the loops connecting the beta-sheets . In the structure of the wild-type holo-azurin from P . aeruginosa the hydrophobic patch is important for the packing of the azurin molecules into dimers which then arrange into tetramers . The completely different packing of the apo-His117Gly mutant can be explained by the disruption of the hydrophobic patch area by the mutation-induced main-chain conformational change of residues 112 to 115 . The structure of apo-His46Gly azurin at 2.5 A resolution is the same as the wild-type structure except for the immediate environment at the site of the mutation . In the His46Gly structure water molecules are found at positions that in the wild-type structure are occupied by the imidazole ring of His46 and the copper ion . The imidazole ring of His117 is shifted by about 1 A towards the surface of the protein, similar to that observed for 50% of the molecules in the wild-type apo-azurin structure . This shift causes a slight rearrangement of the monomers within the tetramer such that one local dyad becomes a crystallographic dyad parallel to the c-axis . This leads to a change in the space group from P2(1)2(1)2(1) to P2(1)2(1)2. FEMS Microbiol Lett, 1997 Feb 15, 147(2), 251 - 7 Pseudomonas aeruginosa rfc genes of serotypes O2 and O5 could complement O-polymerase-deficient semi-rough mutants of either serotype; de Kievit TR et al.; Using a gene-replacement strategy and a mutated copy of the Pseudomonas aeruginosa O5 rfc gene, we were able to generate a rfc mutant in P . aeruginosa serotype O2 . This mutant, which exhibits the semi-rough (SR) LPS phenotype, was used to isolate the O2 rfc gene . Mobilization of the O2 and O5 rfc genes into SR mutants of the heterologous serotype resulted in 'cross-polymerization' of O-repeat units, indicating that the genes are functionally exchangeable . Analysis of the nucleotide sequence of the rfc genes revealed that the two Rfc proteins are identical . The results of this study have enabled us to propose the linkage catalyzed by the O5 O-polymerase enzyme. FEMS Microbiol Lett, 1997 Feb 15, 147(2), 195 - 202 Implication of neutral polysaccharides associated to alginate in inhibition of murine macrophage response to Pseudomonas aeruginosa; Pasquier C et al.; There is evidence that exopolysaccharides (EPS) contribute to the persistence of Pseudomonas aeruginosa in cystic fibrosis lung . However, the relationship between the chemical composition of EPS and the modulation of phagocytic cells is poorly understood . In order to evaluate the role of the chemical composition of EPS in macrophage behavior changes, we pretreated macrophages with characterized EPS and assessed P . aeruginosa phagocytosis and reactive oxygen intermediate (ROI) production . The results showed that alginate and neutral polysaccharides are involved in phagocytic impairment of P . aeruginosa . Moreover, alginates were able to prime macrophages for increased P . aeruginosa-induced macrophage oxidative burst as determined by chemiluminescence . In contrast, neutral polysaccharides are responsible for the decrease of ROI by a scavenging effect evaluated by the xanthine-xanthine oxidase system . This study showed that the content of P . aeruginosa EPS in alginate, but also in neutral polysaccharides, influences the behavior of strains towards phagocytosis and macrophage oxidative burst. Structure, 1997 Feb 15, 5(2), 203 - 16 Crystal structure of the dihaem cytochrome c4 from Pseudomonas stutzeri determined at 2.2A resolution; Kadziola A et al.; Background: . Cytochromes c4 are dihaem cytochromes c found in a variety of bacteria . They are assumed to take part in the electron-transport systems associated with both aerobic and anaerobic respiration . The cytochrome c4 proteins are located in the periplasm, predominantly bound to the inner membrane, and are able to transfer electrons between membrane-bound reduction systems and terminal oxidases . Alignment of cytochrome c4 sequences from three bacteria, Pseudomonas aeruginosa, Pseudomonas stutzeri and Azotobacter vinelandii, suggests that these dihaem proteins are composed of two similar domains . Two distinctly different redox potentials have been measured for the Ps . stutzeri cytochrome c4, however . Results: . The crystal structure of the dihaem cytochrome c4 from Ps . stutzeri has been determined to 2.2A resolution by isomorphous replacement . The model, consisting of two entire cytochrome c4 molecules and 138 water molecules in the asymmetric unit, was refined to an R value of 20.1% for all observations in the resolution range 8-2.2A . The molecule is organized in two cytochrome c-like domains that are related by a pseudo-twofold axis . The symmetry is virtually perfectly close to the twofold axis, which passes through a short hydrogen bond between the two haem propionic acid groups, connecting the redox centre of each domain . This haem-haem interaction is further stabilized by an extensive symmetrical hydrogen-bond network . The twofold symmetry is not present further away from the axis, however, and the cytochrome c4 molecule can be considered to be a dipole with charged residues unevenly distributed between the two domains . The haem environment in the two domains show pronounced differences, mainly on the methionine side of the haem group . Conclusions: . The structure, in conjunction with sequence alignment, suggests that the cytochrome protein has evolved by duplication of a cytochrome c gene . The difference in charge distribution around each haem group in the two domains allows the haem group in the N-terminal domain to be associated with the lower redox potential of 241 mV and the C-terminal haem group with the higher potential of 328 mV . The molecular dipole characteristic of cytochrome c4 is important for its interaction with, and recognition of, its redox partners . In cytochrome c4, the hydrogen-bond network (between residues that are conserved in all known cytochrome c4 subspecies) seems to provide an efficient pathway for an intramolecular electron transfer that can ensure cooperativity between the two redox centres . The C-pyrrole corners of the haem edges are potential sites for external electron exchange. Proc Natl Acad Sci U S A, 1997 Feb 4, 94(3), 967 - 72 Transcriptional activation of mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease; Li JD et al.; An unresolved question in cystic fibrosis (CF) research is how mutations of the CF transmembrane conductance regulator, a Cl ion channel, cause airway mucus obstruction leading to fatal lung disease . Recent evidence has linked the CF transmembrane conductance regulator mutation to the onset and persistence of Pseudomonas aeruginosa infection in the airways, and here we provide evidence directly linking P . aeruginosa infection to mucus overproduction . We show that P . aeruginosa lipopolysaccharide profoundly upregulates transcription of the mucin gene MUC 2 in epithelial cells via inducible enhancer elements and that this effect is blocked by the tyrosine kinase inhibitors genistein and tyr-phostin AG 126 . These findings improve our understanding of CF pathogenesis and suggest that the attenuation of mucin production by lipopolysaccharide antagonists and tyrosine kinase inhibitors could reduce morbidity and mortality in this disease. Clin Infect Dis, 1997 Feb, 24 Suppl 2, S191 - 6 Comparison of antibacterial activities of meropenem and six other antimicrobials against Pseudomonas aeruginosa isolates from North American studies and clinical trials; Iaconis JP et al.; The in vitro activity of meropenem was compared with those of six other antimicrobials against up to 1,182 clinical isolates of Pseudomonas aeruginosa from 16 North American centers by means of standardized controlled methods . Meropenem was the most active drug . These isolates were less frequently resistant to meropenem (4.2%) than to imipenem (12.5%), ceftazidime (15.6%), piperacillin (21%), ciprofloxacin (16%), tobramycin (26%), or gentamicin (29.8%) . Of 147 imipenem-resistant P . aeruginosa isolates, 43.8% were susceptible to meropenem, and 26.9% additional isolates were moderately susceptible to meropenem . Of 49 meropenem-resistant (MIC, > or = 16 micrograms/mL) isolates, 85.7% were also imipenem-resistant, and 24% to 79% were resistant to other antimicrobials . Meropenem MICs were lower than imipenem and ceftazidime MICs for 92 P . aeruginosa isolates from meropenem clinical trials . Carbapenem MICs of > or = 16 micrograms/mL for selected P . aeruginosa isolates from meropenem clinical trials were associated with loss of the approximately 45-kD outer-membrane protein and/or production of type I beta-lactamases . No metallo-beta-lactamases (e.g., "efficient" carbapenemases) were detected. Pharmazie, 1997 Feb, 52(2), 157 - 9 The influence of postantibiotic effects and postantibiotic effects of sub-inhibitory concentrations of quinolones and aminoglycosides on phospholipase C of Pseudomonas aeruginosa; Hybenova D et al.; We have studied the postantibiotic effect (PAE) and postantibiotic sub-MICs effect (PA SME) of ciprofloxacin, pefloxacin, netilmicin and tobramycin on the production of phospholipase C (PLC) in a Pseudomonas aeruginosa strain isolated from clinical material . All antibiotics tested (postantibiotic phase evoked by 2.MIC) reduced the production of PLC, particularly pefloxacin to 13.2% . The PA phase evoked by 4.MIC of quinolones did not cause regrowth of P . aeruginosa and aminoglycosides inhibited PLC, in particular with tobramycin to 16.9% . The effect of PA SME (2.MIC) of netilmicin and tobramycin inhibited PLC more expressively (< 20%) than ciprofloxacin; pefloxacin did not allow regrowth . PA SMEs of the antibiotics tested reduced the production of PLC more effectively than PAE. Infect Control Hosp Epidemiol, 1997 Feb, 18(2), 134 - 6 Nosocomial infections and pseudoinfections from contaminated bronchoscopes: two-year follow up using molecular markers; Blanc DS et al.; During a 6-month period, two genetically distinct clones were isolated from 65% of patients with Pseudomonas aeruginosa isolated from bronchial specimens obtained during or after bronchoscopy . This epidemic was due to contamination of bronchoscopes by washing machines . After reintroduction of manual disinfection of endoscopes, a significant decrease in the incidence of the epidemic clones was observed, but the incidence of nonepidemic P aeruginosa did not change . The distinction between sporadic and epidemic cases was possible only with the use of a molecular typing method (ribotyping). West J Med, 1997 Feb, 166(2), 126 - 8 Soft tissue and bone infections from puncture wounds in children; Laughlin TJ et al.; We report on the prevalence of osteomyelitis, the prevalence of soft tissue infections, and the type and number of pathogens encountered in bone and soft tissue infections caused by puncture wounds in children . In addition, we seek to establish whether shoe gear plays a role in the flora in infected puncture wounds and if laboratory indices are indicative of the presence of infection . The group consisted of 44 nondiabetic children admitted to hospital for puncture wounds of the foot . Cultures were positive for osteomyelitis in 7 patients (16%), all involving the forefoot (P < .04) . The most common pathogen in soft tissue infections was Staphylococcus aureus . The most common pathogen in osteomyelitis was Pseudomonas aeruginosa . There was no significant difference in the prevalence of osteomyelitis and soft tissue infection based on footwear . There were no cases of osteomyelitis encountered among barefoot children (P < .04) . In 10 cases (83%), P aeruginosa infection (both soft tissue and bone) occurred while the patients were wearing tennis shoes (P < .04) . In this study, the leukocyte count (normal in 29 patients {66%}), erythrocyte sedimentation rate (normal in 28 patients {64%}), and temperature (normal in 44 patients {95%}) did not have any predictive value in differentiating soft tissue infection from osteomyelitis in children. J Chemother, 1997 Feb, 9(1), 32 - 7 Effect of antimicrobial agents on the piliation of Pseudomonas aeruginosa and adherence to mouse tracheal epithelium; Yamasaki T et al.; The effect of subminimal inhibitory concentrations (sub-MICs) of antimicrobial agents on the adherence of Pseudomonas aeruginosa (pili+) to trachea mucosal cells, the first stage in the development of P . aeruginosa respiratory tract infections, was investigated . The adherence of P . aeruginosa to the cells of the lower respiratory tract using a model of acid-injured trachea in mice was observed by electron microscopy (transmission and scanning) . When P . aeruginosa was cultured with 1/4 MIC of erythromycin, minocycline, clindamycin, ofloxacin or tobramycin at 37 degrees C for 4 hours, the number of pili was significantly reduced (P < 0.01), together with a significant reduction in the number of adherent bacteria at 1/4 MIC of erythromycin (P < 0.01) . No suppressive effects of piperacillin or ceftazidime were obtained on the piliation and adhesion of P . aeruginosa . These findings indicate that sub-MICs of erythromycin reduce the adherence of P . aeruginosa to the tracheal mucosa, which may prevent the onset of P . aeruginosa respiratory tract infection. Eur J Clin Microbiol Infect Dis, 1997 Feb, 16(2), 159 - 62 In vitro activity of fosfomycin combined with ceftazidime, imipenem, amikacin, and ciprofloxacin against Pseudomonas aeruginosa; Tessier F et al.; The in vitro activity of fosfomycin in combination with ceftazidime, imipenem, amikacin, or ciprofloxacin was studied by an agar plate checkerboard method against 40 clinical isolates of Pseudomonas aeruginosa with various antibiotic resistance profiles . Fosfomycin/ciprofloxacin appeared to be the most effective combination (synergy 15%, addition 80%, indifference 5%), followed by fosfomycin/amikacin (synergy 7.5%, addition 52.5%, indifference 40%), fosfomycin/imipenem (addition 37%, indifference 63%), and fosfomycin/ceftazidime (addition 20%, indifference 80%) . The effects of the combinations were not significantly influenced by the antibiotic susceptibility patterns of the strains. Jpn J Antibiot, 1997 Feb, 50(2), 187 - 94 {Serotypes and drug susceptibility of Pseudomonas aeruginosa isolated from clinical specimens}; Nakae M et al.; Between January, 1982 and December, 1994, 236 Pseudomonas aeruginosa strains were isolated from clinical specimens at our division, and were tested for serotypes and drug-susceptibilities to 15 antibiotics . Serotype G strains were isolated at the highest frequency (32.6%), and followed by strains of serotype B (15.7%), A (11.9%), E (9.3%), I (7.2%), F and M (5.5%), non-typable (5.1%), D (3.4%), H (2.1%), C and K (0.8%) . We examined the changes of isolation frequencies of different serotypes annually . Isolation frequencies of serotypes E and F showed tendency to decrease, whereas serotype I has been isolated increasingly year by year . MIC90's of the 15 antibiotics were as follows, tosufloxacin: 0.78 microgram/ml, biapenem (BIPM) and ofloxacin (OFLX): 3.13 micrograms/ml, imipenem (IPM), ceftazidime, cefozopran, cefsulodin and gentamicin: 6.25 micrograms/ml, aztreonam and amikacin: 12.5 micrograms/ml, piperacillin, cefoperazone and minocycline (MINO): 25 micrograms/ml, fosfomycin: > 100 micrograms/ml and chloramphenicol: > 200 micrograms/ml . MIC90'S of IPM, BIPM, MINO and OFLX increased 4-fold from stage I (1982-1987) through stage III (1992-1994) and the isolation frequency of drug-resistant strains increased year by year . In other words, antibiotic resistant strains appeared increasing with time . No relationship between serotypes and drug-resistance were observed. J Antibiot (Tokyo), 1997 Feb, 50(2), 139 - 42 Affinities of BO-2727 for bacterial penicillin-binding proteins and morphological change of gram-negative rods; Hashizume T et al.; Affinities of BO-2727, a new carbapenem, for penicillin-binding proteins (PBPs) of Escherichia coli . Pseudomonas aeruginosa and Staphylococcus aureus were studied . BO-2727 showed preferential affinity for PBP-2 of E . coli . and induced swollen and ovoid cells, when the organism was exposed to the MIC . In contrast, BO-2727 bound to both PBPs-2 and -3 of P . aeruginosa to a similar extent, and induced short filament cells with bulge . As compared with imipenem and meropenem, there was an observed difference in the affinity, especially for PBP-3, in both organisms; BO-2727 displayed intermediate affinity for PBP-3 between meropenem and imipenem . Studies on the affinity for PBPs of methicillin-susceptible S . aureus showed that the IC50 values for PBP-1 was roughly correlated with the MIC values of carbapenems tested, but those for the PBPs-2 and -3 appeared to be greater than the MIC values . In further studies on the affinity for PBP-2' of methicillin-resistant S . aureus . BO-2727 displayed better binding kinetics than imipenem, which reflected the better activity of BO-2727 than that of imipenem. J Antibiot (Tokyo), 1997 Feb, 50(2), 135 - 8 In vitro and in vivo evaluation of BO-2727 against imipenem- and/or meropenem-resistant Pseudomonas aeruginosa; Shibata K et al.; The in vitro and in vivo activity of BO-2727, a carbapenem antibiotic, against resistant clinical isolates of Pseudomonas aeruginosa was studied . The geometric mean MICs against three groups of clinical isolates resistant to imipenem, meropenem and both carbapenems were 4.28, 4.08 and 5.44 micrograms/ml, respectively . BO-2727 also inhibited multiply antibiotic resistant isolates and laboratory mutants including a nalB-type mutant, which showed resistance to antibiotics such as imipenem, meropenem, ceftazidime, and/or ciprofloxacin, at less than 1.56 micrograms/ml . Overall, BO-2727 was 4-fold more active than biapenem, meropenem, panipenem and imipenem with an MIC90 of less than 6.25 micrograms/ml . The presence of basic amino acids in minimal medium less affected the antipseudomonal activity to a minimal extent, suggesting that BO-2727 has diverse penetration routes through the outer membrane other than OprD channel, which facilitates the diffusion of basic amino acids and carbapenems . The in vitro activity of BO-2727 reflected well in its therapeutic efficacy in experimental systemic infection in mice . These results suggest a possibility for the development of antipseudomonal carbapenems having activity against imipenem- and/or meropenem-resistant P . aeruginosa as well as a broad spectrum encompassing Gram-positive and -negative bacteria. Virchows Arch, 1997 Feb, 430(2), 131 - 7 Bronchiolitis obliterans in ataxia-telangiectasia; Ito M et al.; Pulmonary disease was studied in four patients with ataxia-telangiectasia . Immunodeficiency was characterized by lymphopaenia, hypo-gammaglobulinaemia and decreased T-cell response to phytohaemagglutinin stimulation in mixed lymphocyte cultures . All four patients died from respiratory failure . Autopsy revealed that all four patients suffered from bronchiolitis obliterans in all lobes . Immunohistochemical examination demonstrated expression of MHC class II antigens on bronchiolar epithelium . Pulmonary infections in ataxia-telangiectasia patients included a case of mycoplasma pneumonia, one of cytomegalovirus pneumonia and one of Pseudomonas aeruginosa infection . The aetiology and immunological background of bronchiolitis obliterans are discussed . Bronchiolitis obliterans is a characteristic finding in ataxia-telangiectasia and may be due to the underlying immune deficit. J Antimicrob Chemother, 1997 Feb, 39(2), 273 - 6 Comparative therapeutic efficacy of clinafloxacin in leucopenic mice; Shapiro MA et al.; A cyclophosphamide-induced leucopenic mouse model was used to compare the therapeutic efficacy of clinafloxacin, a fluoroquinolone in clinical trials, with that of ciprofloxacin and imipenem/cilastatin, two clinically relevant standard drugs . Acute systemic infections induced by Escherichia coli, Pseudomonas aeruginosa, a penicillin-resistant Staphylococcus aureus and a methicillin-resistant S . aureus (MRSA) were used to evaluate drug efficacy . Median protective values (PD50) with 95% confidence limits were determined in both leucopenic and normal mice . Results show that clinafloxacin is potentially a useful agent in the treatment of neutropenic patients. J Antimicrob Chemother, 1997 Feb, 39(2), 265 - 7 The effect of antimicrobial agents on the induction of tumour necrosis factor by alveolar macrophages in vitro in response to endotoxin; Nwariaku FE et al.; Alveolar macrophages from New Zealand white rabbits were incubated with twice the MIC of amikacin, ciprofloxacin, aztreonam, ceftazidime and imipenem and exposed to either 10(4), 10(5) or 10(6) cfu/mL live Pseudomonas aeruginosa ATCC 27853 or 0.1, 1 or 10 mg/L purified lipopolysaccharide (LPS) derived from P . aeruginosa to determine the effects of different classes of antimicrobial agent on production of tumour necrosis factor (TNF) . Incubation of macrophages with ciprofloxacin and amikacin resulted in less TNF activity after exposure to live P . aeruginosa than was found for saline, aztreonam, ceftazidime or imipenem (P < 0.05) . However, no significant differences were found between any of the agents after macrophages had been exposed to purified LPS . Different antimicrobial agents therefore appear to exert different effects in vitro on the TNF response of macrophages to bacterial stimulation. J Antimicrob Chemother, 1997 Feb, 39(2), 241 - 6 Chronic osteomyelitis caused by multi-resistant Gram-negative bacteria: evaluation of treatment with newer quinolones after prolonged follow-up; Galanakis N et al.; We evaluated ciprofloxacin, ofloxacin and pefloxacin regimens for the treatment of chronic osteomyelitis due to Gram-negative multiresistant organisms . The study was open, nonrandomized and included 28, 21 and 16 patients, respectively . The 4-fluoroquinolone regimens were 1000 mg, 400 mg and 400-800 mg bd, for a mean duration of 137, 163 and 134 days, respectively . Pseudomonas aeruginosa was the most common pathogen, isolated in 33 individuals . Patients were followed clinically, bacteriologically and radiologically during treatment and for 2-5 years after discontinuation of therapy . Clinical outcome at the end of therapy was successful in 79%, 81% and 75%, improvement occurred in 11%, 10% and 19%, and the failure rate was 11%, 10% and 6%, while 11%, 5% and 6% relapsed, respectively . At the end of follow-up the bacterial eradication rate was 68%, 76% and 69%, respectively . Fluoroquinolone resistance emerged in 18%, 19% and 13% of ciprofloxacin, ofloxacin and pefloxacin recipients, respectively . The newer quinolones were safe and well tolerated and should be considered as the contemporary treatment of choice for chronic Gram-negative osteomyelitis, particularly whenever P . aeruginosa is implicated. J Antimicrob Chemother, 1997 Feb, 39(2), 217 - 22 Effect of 6-fluoro-8-methoxy quinolone (AM-1155) against chronic airway infection with Pseudomonas aeruginosa in a rat model; Sato A et al.; We studied the effect of AM-1155, a newly developed quinolone, against chronic airway infection with Pseudomonas aeruginosa using a previously described rat model . AM-1155 (25 mg/kg) or ciprofloxacin (25 mg/kg) or saline (controls) were injected s.c . for 14 days (from day 4 to day 17) after the inoculation of agar beads containing P . aeruginosa . The number of viable cells of intrapulmonary P . aeruginosa, histological findings of the lungs and immunoglobulin levels of serum and bronchoalveolar lavage fluid were examined in rats 11 and 18 days after the treatment . The findings indicated that the number of viable cells of P . aeruginosa in lungs was significantly decreased in the AM-1155- or ciprofloxacin-treated group compared with the non-treated control group . Histological examination in the non-treated control group showed hyperplasia of bronchus-associated lymphoid tissue as well as cellular infiltration in airways, but not prominently in the AM-1155- or ciprofloxacin-treated group . The IgG and IgA levels in serum and bronchoalveolar lavage fluid were significantly lower in the AM-1155- and ciprofloxacin-treated groups than in the control group . These in-vivo effects of AM-1155 were comparable to those of ciprofloxacin . These findings suggest that treatment with AM-1155 and ciprofloxacin suppressed excessive immune responses, preventing progression of airway damage in the chronic infectious state. J Appl Physiol, 1997 Feb, 82(2), 644 - 51 Protective role of synthetic sialylated oligosaccharide in sepsis-induced acute lung injury; Ridings PC et al.; Proper engagement of leukocyte and endothelial cell selectins with their counterreceptors is an initial step in neutrophil trafficking to sites of inflammation . Certain fucosylated carbohydrate determinants such as sialyl Lewis-x are proposed to act as these counterreceptors . We studied the effects of a synthetic sialyl Lewis-x analog, CY-1503, on the course of hemodynamic derangements and acute lung injury during experimental gram-negative sepsis . Anesthetized ventilated swine were made septic with an infusion of live Pseudomonas aeruginosa . A treatment group received an initial bolus of CY-1503 (60 mg/kg) before sepsis, followed by continuous infusion of CY-1503 (15 mg.kg-1.h-1) . Treatment with CY-1503 did not prevent the development of pulmonary hypertension, systemic hypotension, decline in cardiac output, or severe neutropenia . However, CY-1503 significantly attenuated lung injury, demonstrated by decreased bronchoalveolar lavage protein content and neutrophil influx, lowered lung myeloperoxidase activity, and improved arterial oxygenation . Neutrophils from septic and CY-1503 animals showed significant activation, reflected by upregulated CD18 expression and priming for oxidant burst compared with control animals . This study suggests blockade of selectin interactions as a potential therapeutic intervention in sepsis-induced lung injury. Eur J Immunol, 1997 Feb, 27(2), 347 - 53 Epitope selection in major histocompatibility complex class I-mediated pathway is affected by the intracellular localization of an antigen; Yamazaki H et al.; We analyzed the mode of antigen presentation of an endogenous antigen localized in the cytoplasm or in the mitochondria . Pseudomonas aeruginosa PAO leucine-, isoleucine-, valine-binding protein (LIVAT-BP) encoded by the braC gene was used as a model antigen . Using mouse BALB/3T3 cells, we established two LIVAT-BP transfectants by transfection of a plasmid harboring the intact braC or braC gene fused with the mitochondrial transport signal derived from the yeast COXIV gene . One of the resulting transfectants, BC-15, expressed LIVAT-BP in the cytoplasm, while YZ-710 cells expressed LIVAT-BP in the mitochondria . The splenic effector cells derived from BALB/c mice primed with BC-15 cells exhibited cytotoxic T lymphocyte (CTL) activity against BC-15 cells, but not against YZ-710 cells, whereas splenic effector cells primed with YZ-710 cells exhibited CTL activity against YZ-710 cells, but not against BC-15 cells . Neither group of splenic effector cells showed CTL activity against parental BALB/3T3 cells . These CTL belonged to the CD8+ alphabeta T cell subset . Furthermore, we observed that the CTL activity against t BC-15 cells or YZ-710 cells was blocked with anti-H2-K(d) mAb, but not with anti-H2-D(d) or H2-L(d) mAb . The CTL against BC-15 or YZ-710 cells could kill parental BALB/3T3 cells in the presence of peptides produced by alkali lysis of the LIVAT-BP, suggesting that these CTL indeed recognized the peptide(s) derived from LIVAT-BP . We determined that the epitope for the CTL against BC-15 cells was QYGEGIATEV, corresponding to residues 162-171, and that the epitope recognized by the CTL against YZ-710 cells was GYKLIFRTI, corresponding to residues 123-131 of LIVAT-BP, respectively . Thus, we show here that epitope selection for MHC class I expression is affected by the intracellular localization of the antigenic protein. Semin Oncol, 1997 Feb, 24(1), 132 - 40 Infectious complications of patients undergoing therapy for acute leukemia: current status and future prospects; Chanock SJ et al.; The success of managing the infectious complications of acute leukemia has permitted oncologists to develop new approaches to induction and high-dose therapy . The single most important risk factor for infection is the duration of absolute neutropenia . Historically, most attention was directed towards gram negative aerobes, especially Pseudomonas aeruginosa, but in recent years gram positive bacteria, generally considered to be less virulent, have become the most frequent isolates in most centers . A recent disturbing trend is the isolation of vancomycin-resistant enterococci . A recent controversy has been whether to use empirical vancomycin; the Centers for Disease Control has issued a formal recommendation discouraging empirical vancomycin in the febrile neutropenic patient . Empirical monotherapy has replaced combination therapy in many institutions except where there has been an increase in resistant isolates . In patients who remain profoundly neutropenic, fungal infections represent a serious source of secondary infection, especially species of Candida and Aspergillus . Recently lipid-based formulations of amphotericin B have offered reduced nephrotoxicity . Less toxic antifungals, the azoles, which include fluconazole and itraconazole, offer an attractive alternative to amphotericin B . New patterns of invasive mycoses have emerged, as for example hepatosplenic candidiasis, presenting new problems in diagnosis and therapy . The successful management of virus infections with herpes simplex, cytomegalovirus, varicella zoster, and Epstein Barr virus is based on early recognition and careful attention to prevention. Microbiology, 1997 Feb, 143 ( Pt 2), 641 - 52 A novel gene, algK, from the alginate biosynthesis cluster of Pseudomonas aeruginosa; Aarons SJ et al.; Colonization of the cystic fibrosis lung by Pseudomonas aeruginosa is greatly facilitated by the production of an exopolysaccharide called alginate . Many of the enzymes involved in alginate biosynthesis are clustered in an operon at 34 min on the P . aeruginosa chromosome . This paper reports the nucleotide sequence of a previously uncharacterized gene, algK, which lies between the alg44 and algE genes of the operon . DNA sequencing data for algK predicted a protein product of approximately 52.5 kDa which contains a putative 27 amino acid N-terminal signal sequence and a consensus cleavage and lipid attachment site for signal peptidase II . Expression of algK using either T7 or tac promoter expression systems, in vivo labelling studies with {35S}methionine, indicated that algK encodes a polypeptide of approximately 53 kDa which is processed to a mature protein of approximately 50 kDa when expressed in Escherichia coli or P . aeruginosa, in agreement with the nucleotide sequence analysis . Results from an algK-beta-lactamase fusion survey support this interpretation and also provide evidence that mature AlgK is entirely periplasmic and is probably membrane-anchored. J Med Genet, 1997 Feb, 34(2), 89 - 91 Genotype-phenotype relationship in 12 patients carrying cystic fibrosis mutation R334W; Antinolo G et al.; We present a phenotype-genotype correlation analysis in 12 patients with cystic fibrosis (CF) carrying the mutation R334W in the CFTR gene . The clinical data obtained for this group were compared with the clinical data of deltaF508/deltaF508 patients . Current age and age at diagnosis were significantly higher in the R334W mutation group (p=0.028 and p=0.0001) . We found a lower rate of Pseudomonas aeruginosa colonisation in patients carrying the R334W mutation, although the difference was not found to be statistically significant . However, we found a statistically significant higher age of onset of Pseudomonas aeruginosa colonisation (p=0.0036) in the group of patients with the R334W mutation . Thirty three percent of R334W patients were pancreatic insufficient, significantly lower than the deltaF508/deltaF508 patients (p=0.004) . We also found that the weight expressed as a percentage of ideal weight for height was significantly higher in patients with the R334W mutation (p=0.0028). Appl Environ Microbiol, 1997 Feb, 63(2), 380 - 6 Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium; Wang SL et al.; Two extracellular chitinases (FI and FII) were purified from the culture supernatant of Pseudomonas aeruginosa K-187 . The molecular weights of FI and FII were 30,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 60,000 and 30,000, respectively, by gel filtration . The pIs for FI and FII were 5.2 and 4.8, respectively . The optimum pH, optimum temperature, pH stability, and thermal stability of FI were pH 8, 50 degrees C, pH 6 to 9, and 50 degrees C; those of FII were pH 7, 40 degrees C, pH 5 to 10, and 60 degrees C . The activities of both enzymes were activated by Cu2+; strongly inhibited by Mn2+, Mg2+, and Zn2+; and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol . Both chitinases showed lysozyme activity . The purified enzymes had antibacterial and cell lysis activities with many kinds of bacteria . This is the first report of a bifunctional chitinase/lysozyme from a prokaryote. Antimicrob Agents Chemother, 1997 Feb, 41(2), 308 - 13 Immunomodulating effect of fosfomycin on gut-derived sepsis caused by Pseudomonas aeruginosa in mice; Matsumoto T et al.; We evaluated the protective effect of fosfomycin (FOM) and an enantiomer of fosfomycin {FOM (+); an isomer of FOM with no bactericidal activity} on murine gut-derived sepsis caused by Pseudomonas aeruginosa . Endogenous bacteremia was induced by administering cyclophosphamide (CY) and ampicillin to specific-pathogen-free mice fed P . aeruginosa . Treatment of mice with FOM at 250 mg/kg of body weight per day twice a day after the second CY administration significantly increased the survival rate compared to that for control mice treated with saline . Treatment with FOM (+) at 20 and 100 mg/kg also significantly increased the survival rate (from 30% for control mice to 80% for treated mice) . The bacterial counts in the liver and blood were both significantly lower in FOM(+)-treated mice in comparison with those in liver and blood of saline-treated control mice . FOM(+) administration affected neither the bacterial colonization in the intestinal tract nor the leukocyte counts in the peripheral blood of the mice . After intravascular inoculation of P . aeruginosa, treatment of mice with FOM (+) did not enhance bacterial clearance from the blood of mice pretreated or not enhance bacterial clearance from the blood of mice pretreated or not pretreated with CY, FOM(+) significantly suppressed tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 levels in the serum of mice after gut-derived sepsis . These results indicate that both FOM and FOM(+) have protective effects against P . aeruginosa bacteremia, despite a lack of specific activity of FOM(+), and suggest that FOM may possess immunomodulating activity and that it induces a protective effect . The protective mechanism is speculated to be that FOM modulates the vivo production of inflammatory cytokines. Infect Immun, 1997 Feb, 65(2), 579 - 86 Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S; Fleiszig SM et al.; Pseudomonas aeruginosa, an opportunistic pathogen, is capable of establishing both chronic and acute infections in compromised hosts . Previous studies indicated that P . aeruginosa displays either a cytotoxic or an invasive phenotype in corneal epithelial cells . In this study, we used polarized MDCK cells for in vitro infection studies and confirmed that P . aeruginosa isolates can be broadly differentiated into two groups, expressing either a cytotoxic or an invasive phenotype . In vivo infection studies were performed to determine if cytotoxic and invasive strains displayed differential pathology . Invasion was assayed in vivo by in situ infection of mouse tracheal tissue followed by electron microscopy . Both cytotoxic and invasive strains entered mouse tracheal cells in situ; however, more necrosis was associated with the cytotoxic strain . In an acute lung infection model in rats, cytotoxic strains were found to damage lung epithelium more than invasive strains during the short infection period of this assay . The expression of cytotoxicity requires a functional exsA allele . In the strains tested, the ability to invade epithelial cells in vitro appears to be independent of exsA expression . Since ExsA is a transcriptional regulator of the exoenzyme S regulon, chromosomal preparations from invasive and cytotoxic strains were screened for their complement of exoenzyme S structural genes, exoS, encoding the 49-kDa ADP-ribosyltransferase (ExoS), and exoT, encoding the 53-kDa form of the enzyme (Exo53) . Invasive strains possess both exoS and exoT, while cytotoxic strains appear to have lost exoS and retained exoT . These data indicate that the expression of cytotoxicity may be linked to the expression of Exo53, deletion of exoS and perhaps other linked loci, or expression of other ExsA-dependent virulence determinants . In the absence of a functional cytotoxicity pathway (exsA::omega strains), invasion of eukaryotic cells is detectable. Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 439 - 42 Polyphosphate kinase as a nucleoside diphosphate kinase in Escherichia coli and Pseudomonas aeruginosa; Kuroda A et al.; Generation of a wide variety of nucleoside (and deoxynucleoside) triphosphates (NTPs) from their cognate nucleoside diphosphates (NDPs) is of critical importance in virtually every aspect of cellular life . Their function is fulfilled largely by the ubiquitous and potent nucleoside diphosphate kinase (NDK), most commonly using ATP as the donor . Considerable interest is attached to the consequence to a cell in which the NDK activity becomes deficient or over-abundant . We have discovered an additional and possibly auxiliary NDK-like activity in the capacity of polyphosphate kinase (PPK) to use inorganic polyphosphate as the donor in place of ATP, thereby converting GDP and other NDPs to NTPs . This reaction was observed with the PPK activity present in crude membrane fractions from Escherichia coli and Pseudomonas aeruginosa as well as with the purified PPK from E . coli; the activity was absent from the membrane fractions obtained from E . coli mutants lacking the ppk gene . The order of substrate specificity for PPK was: ADP > GDP > UDP, CDP; activity with ADP was 2-60 times greater than with GDP, depending on the reaction condition . Although the transfer of a phosphate from polyphosphate to GDP by PPK to produce GTP was the predominant reaction, the enzyme also transferred a pyrophosphate group to GDP to form the linear guanosine 5' tetraphosphate. FEMS Microbiol Lett, 1997 Jan 15, 146(2), 311 - 8 Dissection of the promoter/operator region and evaluation of N-acylhomoserine lactone mediated transcriptional regulation of elastase expression in Pseudomonas aeruginosa; Fukushima J et al.; In Pseudomonas aeruginosa, expression of the lasB gene which codes for the metalloprotease, elastase, depends on small diffusible N-acylhomoserine lactones . lasB expression is regulated through the interactions of N-3-oxododecanoyl-L-homoserine lactone and N-butanoyl-L-homoserine lactone with the transcriptional activators LasR and VsmR(RhlR), respectively . To investigate lasB expression further, we first located the transcriptional start site to a position 141 bp upstream from the translational start site . Using this information, we constructed a series of plasmids containing consecutive 5' deletions of the upstream region of lasB fused to a promoterless chloramphenicol acetyltransferase reporter gene . The results obtained indicate that three regions are required for efficient transcription of lasB; a 35 bp palindromic sequence located at +26 to +60 bp upstream from the translation start site, and two regions located upstream of the transcription start site, at -135 to -85 bp and -63 to -26 bp, respectively . Deletion of the latter region results in the loss of both N-butanoyl-L-homoserine lactone- and N-3-oxododecanoyl-L-homoserine lactone-mediated stimulation of lasB expression and provides further support for the role of this operator site as a target for either or both LasR and VsmR. J Clin Invest, 1997 Jan 15, 99(2), 325 - 35 Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-alpha-dependent mechanism; Rezaiguia S et al.; To study the rate and regulation of alveolar fluid clearance in acute pneumonia, we created a model of Pseudomonas aeruginosa pneumonia in rats . To measure alveolar liquid and protein clearance, we instilled into the airspaces a 5% bovine albumin solution with 1.5 microCi of 125I-human albumin, 24 h after intratracheal instillation of bacteria . The concentration of unlabeled and labeled protein in the distal airspaces over 1 h was used as an index of net alveolar fluid clearance . Since there was histologic evidence of alveolar epithelial injury, several methods were used to measure alveolar fluid clearance, including the use of experiments in rats with blood flow and the use of experiments in rats without blood flow, so that movement across the epithelial barrier would be minimized in the latter group . The results with each method were identical . We found that P . aeruginosa pneumonia increased alveolar liquid clearance over 1 h by 48% in studies with blood flow, and by 43% in rats without blood flow, compared with respective controls (P < 0.05) . In both studies, this increase was inhibited with amiloride . However, propranolol had no inhibitory effect, thus ruling out a catecholamine-dependent mechanism to explain the increase in alveolar fluid clearance . An antitumor necrosis factor-alpha neutralizing antibody, instilled into the lung 5 min before bacteria, prevented the increase in alveolar liquid clearance in rats with pneumonia (P < 0.05) . Also, TNFalpha (5 microg) instilled in normal rats increased alveolar liquid clearance by 43% over 1 h compared with control rats (P < 0.05) . In normal rats instilled with TNFalpha, propranolol had no inhibitory effect . In conclusion, gram-negative pneumonia markedly upregulates net alveolar epithelial fluid clearance, in part by a TNFalpha-dependent mechanism . This finding provides a novel mechanism for the upregulation of alveolar epithelial sodium and fluid transport from the distal airspaces of the lung. Acta Microbiol Pol, 1997, 46(1), 37 - 43 A study of purified pyorubrin produced by local Pseudomonas aeruginosa; Kandela SA et al.; A study of more than one thousand strains of local P . aeruginosa was performed . These include the strains production of various pigments, methods of extraction and purification, the pigments activity and characteristics . Lately, we have reported a comprehensive study about the purified blue pyocyanine pigment . This paper displays a parallel study of a number of physical and chemical properties of the purified pink pigment, the pyorubrin, that is produced by our local collection of P . aeruginosa strains . This characteristics is useful for production of an active pigment purpose and is necessary in the pigment use as antibiotic, in addition of importance in developing the methods of strains identification . Such studies are important due to the biological significance of pyorubrin activity against other bacteria and in comparison with some known antibiotics; stability tests and MIC (minimum inhibitory concentration) measurements indicate that found isolates were of animal and food origin mainly