Pseudomonas aeruginosa

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.

Zh Mikrobiol Epidemiol Immunobiol, 1997 Jan-Feb, (1), 6 - 9
{The physicochemical characteristics of ribosomal fractions from Pseudomonas aeruginosa obtained by precipitation with polyethylene glycol 6000}; Egorova TP et al.; Ribosomal fractions containing up to 72% of ribosomal material and 25% of sugars (among them, about 6% of hexose) were isolated from P.aeruginosa cells, immunotypes F-1, 2, 6 and 7, by precipitation with polyethylene glycol 6000 . Lipopolysaccharide, determined in the test for ketodesoxyoctanoic acid, was not detected in these fractions, but, as determined in the passive hemagglutination test, the content of O-antigen in the preparations was 3-25% . O-antigen and ribosome present in the fractions formed a complex, disintegrating after treatment with trilon B.

Zh Mikrobiol Epidemiol Immunobiol, 1997 Jan-Feb, (1), 54 - 8
{The comparative characteristics of the effect of immunomodulators on the indices of body nonspecific protection and on the structure of the populations of the causative agents in mixed infections}; Shatalova EV et al.; A sharp decrease in the characteristics of nonspecific protection and humoral immunity in mixed infections (Pseudomonas aeruginosa + fungi of the genus Candida) in cases of burn traumas was established . Under these conditions an increase (up to 100%) in the number clones with the signs of pathogenicity and multiple medicinal resistance in the populations of infective agents, accompanied by a high death rate among the animals (up to 60%), was observed in the dynamics of the infectious process . The use of immunomodulators (tactivin and sodium nucleinate + lidocaine) contributed to the restoration of the phagocytic function of peripheral blood lymphocytes and humoral immunity, as well as to a decrease in the death rate of the animals . At the same time a considerable decrease in the number of clones with the signs of pathogenicity was observed in the populations in infective agents, isolated from the animals in the process of treatment.

Rev Pneumol Clin, 1997, 53(2), 91 - 7
{Aerosol therapy and cystic fibrosis: a national survey}; Huet F et al.; Aerosol therapy in cystic fibrosis is indicated in order to administer active agents directly into the diseased organ . The practical application of this technique often remains a question of personal experience rather than rigorous and validated schemes . In order to determine the experience of those using this technique, a questionnaire was prepared by the health care centers certified by the French association against cystic fibrosis (65 centers) . There were 53 responses (82%) covering 3400 patients . Two-thirds of these patients (2250) used nebulizers . Drugs used by order of frequency were: rhDNase (52 centers), antibiotics (51), conventional fluidifying agents (17), isotonic saline solution (14) . Bronchodilators were used as sprays or inhalation chambers . Antibiotics most frequently prescribed were: colimycin (51 centers, doses 500.000 to 3 MIU per aerosol), tobramycin (44 centers, 25 to 600 mg per aerosol), amikacine (7 centers, 150 mg to 1.5 g per aerosol) . Indications were mainly chronic colonization with Pseudomonas aeruginosa (39/53 centers) and episodes of bronchial superinfection with Pseudomonas aeruginosa (18/53 centers) . Equipment maintenance appeared as the main problem in most centers (daily or more cleaning with antiseptic solutions in 46/53 centers) . The differences in indications and dosages prescribed justify further prospective randomized studies to optimize treatment.

Adv Exp Med Biol, 1997, 419, 71 - 82
ADP-ribosylation and early transcription regulation by bacteriophage T4; Wilkens K et al.; Bacteriophage T4 codes at least for two ADP-ribosylating activities, the 76 kDa Alt and the 24 kDa Mod gene products . The main target for both enzymes is the host RNA polymerase . We cloned and sequenced the alt gene and overexpressed the corresponding enzyme . The recombinant protein shows ADP-ribosylating activities in vitro, as had been described earlier for the native enzyme isolated from phage heads . The native as well as the recombinant protein ADP-ribosylate the alpha-subunit of RNA polymerase, but also subunits beta, beta' and sigma 70 and perform an autoribosylation reaction . Taking advantage of the pKWIII test system, constructed to measure promoter strengths in vivo, it was found that ADP-ribosylation of RNA polymerase leads to an increase of transcription from T4 early promoters up to a factor of two . In an infected host cell this should cause an enhanced expression of T4 genes . Depending on whether RNA polymerase was ADP-ribosylated or not, it initiated transcription at T4 promoters with different sequence characteristics: unribosylated RNA polymerase recognizes the early T4 promoters by an extended -10 region, whereas the ribosylated enzyme selects for T4 early promoters with an extended T4-specific and highly conserved -35 region . These results may reflect how the virus, step by step imposes its genetic program on the host cell, and in part they give a rationale for the extension of the consensus sequence observed with these promoters . We also sequenced the genomic region of the T4 mod gene and found two open reading frames coding both for proteins of approximately 24 kDa . Up to now none of the reading frames could be cloned into E . coli in an active form, making it highly probable that the ADP-ribosylation pattern inflicted by gene product Mod on host RNA polymerase is deleterious to these bacteria . Comparisons of the amino acid sequences showed significant homologies among the two reading frames . Computer analysis reveals that both Mod sequences and also the sequence of the Alt protein exhibit a structural concordance with the catalytic domains of other prokaryotic ADP-mono-ribosyltransferases such as the Pseudomonas aeruginosa exotoxin A, the cholera labile enterotoxin, the diphteria toxin, the heat labile enterotoxin A of E . coli, and pertussis toxin . We present a detailed model for T4 transcription regulation.

J Basic Microbiol, 1997, 37(2), 115 - 28
Localization of alanyl aminopeptidase and leucyl aminopeptidase in cells of Pseudomonas aeruginosa by application of different methods for periplasm release; Jensch T et al.; Various methods for the isolation of periplasm were examined and compared with regard to the complete release of known periplasmic marker enzymes and the contamination of the periplasm by cytosol for Pseudomonas aeruginosa PAO1 as a significant Gram-negative test strain . The aim of the investigations was to clarify the exact localization of alanyl aminopeptidase (AAP) and leucyl aminopeptidase (LAP) of this microorganism and to evaluate these methods . The osmotic shock of NOSSAL and HEPPEL (1996) was the most effective method with the lowest contamination by the cytosolic marker enzyme malic enzyme, but some proteins, which are located near the inner side of the cytoplasmic membrane, can be released additionally into the periplasm . All other procedures like chloroform or polymyxin treatment, the magnesium chloride washing of intact bacteria and spheroblasting by lysozyme in the presence of EDTA or magnesium chloride resulted only in a partial, sometimes only very low release of periplasm . The periplasmic enzymes are bound either more by hydrophobic or more by ionic interactions to the cell envelope and show a different behaviour with the different releasing agents . These methods are useful for a further differentiation between really periplasmic protein, and those proteins, which were false positive found in periplasm as a result of the osmotic shock . Our results show that AAP from Pseudomonas aeruginosa is a periplasmic enzyme with hydrophobic interactions to the cytoplasmic membrane, corresponding to the early results of LAZDUNSKI and MURGIER for Escherichia coli (LAZDUNSKI et al . 1975a and b, MURGIER et al . 1977), and LAP is cytosolic, but located near the cytoplasmic membrane . The AAP is not a real amphipatic membrane protein, as could be demonstrated by phase separation experiments with Triton X-114.

Retina, 1997, 17(2), 139 - 45
Experimental pseudomonal posttraumatic endophthalmitis in a swine model . Treatment with ceftazidime, amikacin, and imipenem; Alfaro DV 3rd et al.; PURPOSE: To compare the intravitreal efficacy of three separately administered antibiotics (imipenem, ceftazidime, and amikacin) in limiting the intraocular inflammation and tissue destruction caused by posttraumatic pseudomonal endophthalmitis . METHODS: Thirty-three Yorkshire pigs each received a surgically induced scleral injury to the right eye . After repair, each eye was injected with 22,000 colony-forming units of live Pseudomonas aeruginosa . Pigs then were randomly grouped into a natural-history-of-infection group in which no treatment was given (n = 9) or into groups treated with the following: intravitreal imipenem (n = 6), ceftazidime (n = 6), amikacin (n = 6), or normal saline (n = 6) . Pigs then were observed clinically for 18-24 hours after surgery and enucleated for histopathologic examination . RESULTS: Clinical examinations revealed significantly less posterior segment inflammation in pigs treated with amikacin and imipenem than in pigs in the natural history or saline control groups, based on the Wilcoxon rank sum test (P < .05) . Histopathologic examinations showed similar results, with less intraocular inflammation and retinal destruction in pigs treated with amikacin and imipenem, whereas the inflammation in pigs treated with ceftazidime did not differ significantly from that in control pigs . CONCLUSION: Intravitreal antibiotic treatment with imipenem or amikacin appears to limit intraocular inflammation and retinal tissue damage when given early in the course of posttraumatic pseudomonal endopthalmitis . Results with ceftazidime are less conclusive.

Kansenshogaku Zasshi, 1997 Jan, 71(1), 72 - 82
{Optimal antibacterial chemotherapy for infectious diseases associated with hematological malignancies}; Sakamoto M; The status quo of infectious diseases associated with hematological malignancies was examined for clinical consideration . In addition, in vitro antibacterial activities and combination effects were also examined by means of various isolated strains derived from sepsis . In clinical practice, 76% of the fevers in patients with hematological malignancies was attributed to infectious diseases mainly involving "fever with granulocytopenia", sepsis, and pneumonia . The detection rate of causal pathogen remained at a low level and more than half of the causes of death were infectious diseases . In the in vitro examination, a favorable antibacterial effect was noted with vancomycin (VCM) for methicillin resistant Staphylococcus aureus (MRSA) and Enterococcus spp., imipenem (IPM) for methicillin sensitive S . aureus (MSSA), and ciprofloxacin, amikacin (AMK), and IPM for Pseudomonas aeruginosa and intestinal flora . But its clinical effect is not enough . Synergic and/or additive effect can be expected by combining IPM with VCM for MRSA and Enterococcus spp., and IPM with AMK for P . aeruginosa . These combination therapies were considered to be optimal as antibacterial chemotherapy for infectious diseases associated with hematological malignancies.

Crit Rev Microbiol, 1997, 23(1), 47 - 75
Pseudomonas aeruginosa: assessment of risk from drinking water; Hardalo C et al.; Pseudomonas aeruginosa is an ubiquitous environmental bacterium . It can be recovered, often in high numbers, in common food, especially vegetables . Moreover, it can be recovered in low numbers in drinking water . A small percentage of clones of P . aeruginosa possesses the required number of virulence factors to cause infection . However, P . aeruginosa will not proliferate on normal tissue but requires previously organs . Further narrowing the risk to human health is that only certain specific hosts are at risk, including patients with profound neutropenia, cystic fibrosis, severe burns, and those subject to foreign device installation . Other than these very well-defined groups, the general population is refractory to infection with P . aeruginosa . Because of its ubiquitous nature, it is not only not practical to eliminate P . aeruginosa from our food and drinking water, but attempts to do so would produce disinfection byproducts more hazardous than the species itself . Moreover, because there is no readily available sensitive and specific means to detect and identify P . aeruginosa available in the field, any potential regulation governing its control would not have a defined laboratory test measure of outcome . Accordingly, attempts to regulate P . aeruginosa in drinking water would not yield public health protection benefits and could, in fact, be counterproductive in this regard.

Am J Rhinol, 1997 Jan-Feb, 11(1), 15 - 25
Gram negative sinusitis: a bacteriologic and histologic study in rabbits; Bolger WE et al.; Recent investigations of chronic sinusitis that is "recalcitrant" to traditional medical and surgical therapy indicate that gram negative bacteria are frequently involved, most commonly Pseudomonas aeruginosa . Analysis of infection-induced histopathologic changes in the underlying sinus mucosa may provide important insight into the recalcitrant nature of these infections . Therefore, the aim of this investigation is to experimentally induce sinus infection in the rabbit with Pseudomonas aeruginosa, a bacterium commonly associated with "recalcitrant sinusitis," and evaluate the histopathologic findings . A unilateral maxillary sinusitis was induced in 33 New Zealand white rabbits . Histologic analysis at 4, 14, 21, and 28 days revealed a moderate inflammation that persisted throughout the study period . Initial changes included edema, loss of submucosal glands, and ulceration . Fibroplasia and bone remodeling were evident throughout the study . Epithelial plaque formation occurred early in the infection, whereas goblet cell formation was a later change . Experimental sinusitis induced by Pseudomonas aeruginosa causes an intense transmucosal injury . The histopathologic injury and response to Pseudomonas aeruginosa appears to be more intense than that noted on previous investigations of experimental sinusitis using other bacteria . The significant histopathologic changes noted could explain the recalcitrant nature of gram negative sinusitis observed clinically in patients.

Mol Microbiol, 1997 Jan, 23(2), 345 - 54
Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa; Kohler T et al.; Antibiotic-resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents . These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50 kDa, which was shown by Western blot analysis to correspond to OprN . A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB-oprM efflux operon . Antibiotic-accumulation studies indicated that OprN was part of an energy-dependent antibiotic-efflux system . Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB-oprM and mexCD-oprJ efflux operons of P . aeruginosa . The ORFs were designated MexE, MexF and OprN . Mutation of the mexE gene eliminated the multidrug-resistance phenotype in an OprN-overexpressing strain, but did not affect the susceptibility profile of the wild-type strain . Expression of the mexEF-oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators . The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild-type strain but not onto the mexE mutant . Evidence is provided to show that the mexEF-oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P . aeruginosa.

Acta Otolaryngol, 1997 Jan, 117(1), 73 - 9
Effect of Pseudomonas aeruginosa exotoxin A on inner ear function; Stenqvist M et al.; Electrophysiological changes were studied in the albino rat following instillation of Pseudomonas aeruginosa exotoxin A into the middle ear cavity through the tympanic membrane . Hearing threshold was measured by a burst-elicited, frequency-specific auditory brainstem response (ABR) technique prior to exposure, then 24 and 48 h, 5 days, 2 and 4 weeks after the toxin instillation . A single dose (1 microgram/20 microliters) of Pseudomonas aeruginosa exotoxin A raised the ABR threshold over the whole frequency range, by 5-25 dB, particularly in the high tones . All threshold shifts were of combined conductive and cochlear type, reversible, with deterioration starting at 24-48 h and recovery at 2-4 weeks . Effusion of serous fluid occurred at 24 or 48 h, resulting in conductive hearing loss . Latency/intensity curves revealed a cochlear component in addition to conductive hearing loss . Morphological examination by SEM showed slight and inconsistent derangement of OHCs . It is concluded that Pseudomonas aeruginosa exotoxin A causes middle ear inflammation, facilitating penetration to the inner ear and that this toxin also reversibly affects cochlear function.

J Foot Ankle Surg, 1997 Jan-Feb, 36(1), 36 - 43
Cost effectiveness of magnetic resonance imaging in diagnosing Pseudomonas aeruginosa infection after puncture wound; Lau LS et al.; This study was performed to determine the cost effectiveness of radiologic imaging studies in diagnosing Pseudomonas aeruginosa infections after puncture wounds and the respective comparison of imaging and scintigraphy . Using retrospective medical record review, we studied 12 patients with culture-proven Pseudomonas infections of bone, cartilage or joint, or soft tissues . Attention was paid to radiographic presentation, scintigraphic studies, and magnetic resonance imaging results . All available imaging studies were reviewed . A survey of the costs of each imaging study was conducted . All patients underwent surgery . Three patients had magnetic resonance imaging studies that provided definitive diagnosis of osteomyelitis and precise localization of involved bones, soft tissue edema, and periosteal abscess . Eight patients had bone scans, one of which was consistent with cellulitis; seven, with osteomyelitis; and two with no abnormalities . All patients had radiographs of the involved foot . Case 4 had three series of radiographs . Nine of the twelve patients (75%) had normal findings on radiographs, with only soft tissue swelling or osteoporosis . Five patients had evidence of osteomyelitis . Magnetic resonance imaging is a cost-effective method in diagnosing Pseudomonas osteomyelitis . Early use of magnetic resonance imaging can provide definitive diagnosis and more precise anatomic localization necessary for surgical intervention.

J Cutan Pathol, 1997 Jan, 24(1), 56 - 60
Superinfected cutaneous angiosarcoma: a highly malignant neoplasm simulating an inflammatory process; Diaz-Cascajo C et al.; This report describes a patient with a poorly differentiated cutaneous angiosarcoma (CA) of the face superinfected with pseudomonas aeruginosa . Neoplastic cells were positive for CD-34, CD-31 and vimentin, whereas they failed to express other vascular markers such as Factor VIII and Ulex europeaus lectin . The tumor spread rapidly through the skin and the superficial soft tissue before metastasizing . The patient died of disease 6 months after histopathological diagnosis . An autopsy revealed widespread metastases in the lung and the liver . The aim of this report is to call attention to some circumstances in which CA may masquerade as an inflammatory process, delaying the right diagnosis with serious consequences for the patient.

Microbiology, 1997 Jan, 143 ( Pt 1), 35 - 43
Use of siderophores to type pseudomonads: the three Pseudomonas aeruginosa pyoverdine systems; Meyer JM et al.; Eighty-eight Pseudomonas aeruginosa isolates, most of them from the Collection of Bacterial Strains of the Institut Pasteur, Paris, were analysed for their pyoverdine-mediated iron incorporation system by different methods, including pyoverdine isoelectrofocusing analysis, pyoverdine-mediated growth stimulation, immunoblot detection of (ferri)pyoverdine outer-membrane receptor and pyoverdine-facilitated iron uptake . The same grouping of the strains was reached by each of these methods, resulting in the classification of the P . aeruginosa isolates, even those which were devoid of pyoverdine production, into three different siderophore types . Forty-two percent of the strains were identified with the type-strain P . aeruginosa ATCC 15,692 (group I), 42% were identical with the second type-strain P . aeruginosa ATCC 27,853 (group II) and 16% reacted identically with the clinical isolate P . aeruginosa Pa6, whose pyoverdine was recognized in this study to be identical in structure to the pyoverdine produced by a natural isolate, P . aeruginosa strain R . No new pyoverdine species was detected among these strains.

Surg Today, 1997, 27(2), 154 - 8
Effects of granulocyte colony-stimulating factor on bacterial translocation due to burn wound sepsis; Yalcin O et al.; The presence of certain defects in both cellular and humoral immunity after thermal injury has been established . Likewise, the translocation of enteric bacteria to the mesenteric lymph nodes and to distant organs has also been observed following serious thermal injury . The effects of granulocyte colony-stimulating factor (G-CSF) on bacterial translocation, the small bowel mucosa, and cecal bacterial content were investigated in a rat model of burn wound sepsis in which albino Wistar rats were scalded over 30% of their bodies, after which the lesions were infected by 1 x 10(8) colony-forming units (cfu) Pseudomonas aeruginosa . The control group was treated with 5% dextrose solution subcutaneously starting 2 days preburn, while the treatment group received 100 microg/kg human G-CSF subcutaneously . On the 4th day post burn all animals were killed to examine the bowel and culture of the mesenteric lymph nodes (MLN), livers, and spleens . No significant differences were observed between the groups regarding the cecal bacterial content and small bowel; however, a difference was seen in the ratio of translocation in the MLN liver and spleen and quantitative MLN cultures . Based on these findings, G-CSF was thus found to be significantly effective in reducing bacterial translocation due to burn wound sepsis.

Bone Marrow Transplant, 1997 Jan, 19(1), 55 - 9
Paranasal sinusitis following allogeneic bone marrow transplant; Savage DG et al.; In attempt to identify major clinical features of paranasal sinusitis following allogeneic BMT, we reviewed 44 consecutive cases diagnosed at the Hammersmith Hospital between August 1993 and December 1995 . All patients had symptoms and signs characteristic of sinusitis . Plain radiographs and/or CT scans revealed fluid levels in 86.4% of patients, opacification in 9.1%, and marked mucosal thickening in 4.5% . Two-thirds of patients were diagnosed within 120 days of BMT . The WBC was less than 1 x 10(9)/1 in 16.3% of patients, the neutrophil count was less than 0.5 x 10(9)/1 in 18.6%, and serum immunoglobulins were depressed (< 6.7 g/l) in 40.6% . Grade III-IV acute GVHD was present in 25.6% of patients and grade I-II in 66.7%; 68.6% developed chronic GVHD . There were 70.5% of patients receiving corticosteroids . Specific pathogens could not be identified in most cases . Pneumonia was present in 10 patients, seven of whom had Aspergillus species identified by bronchoalveolar lavage . Parainfluenza virus was isolated in three patients and Pseudomonas aeruginosa in two . Although all patients received antimicrobial therapy, 70.5% developed chronic sinusitis . Fatal complications did not occur . In 94 consecutive patients receiving allografts for CML during the period of study, WBC and neutrophil counts were lower 3 months post-BMT in patients who developed sinusitis (P < 0.02) . Patients receiving higher doses of total body irradiation (13.2 and 14.4 Gy) had a greater probability of developing sinusitis (P = 0.023) . Sinusitis occurred in only one of 37 patients receiving autologous transplants in the same period . Sinusitis is common following allogeneic BMT . Leukopenia is often present, but microbiological diagnosis is difficult, and progression to chronic sinusitis common.

FEMS Immunol Med Microbiol, 1997 Jan, 17(1), 37 - 47
Arbitrary primed PCR fingerprinting and serotyping of clinical Pseudomonas aeruginosa strains; Hernandez J et al.; Arbitrary primed PCR (AP-PCR) analysis was compared with serotyping as a means of high-resolution typing of Pseudomonas aeruginosa . Seventy-four isolates from 3 different hospitals and 18 reference strains were studied . Serotyping provided good index of discrimination, although eleven isolates could not be serotyped . Genomic DNA was amplified with a single 10 nucleotide primer (sequence 5'-AGG GGT CTT G-3') . The strains were genetically diverse and 61 different AP-PCR profiles of 2-7 bands between 0.3 and 2.4 kb were obtained . AP-PCR profiles were not consistently associated with serotypes, but they clearly subtyped strains of the same serotype . Numerical analysis of AP-PCR patterns defined 7 groups at the 55% similarity level, and identified predominant strains in each hospital . The results show that AP-PCR analysis provides a simple and practical approach to typing P . aeruginosa that is more discriminatory than traditional serotyping scheme . We suggest that maximum discrimination can be achieved by a combination of both methods.

Arch Surg, 1997 Jan, 132(1), 65 - 75
Delayed administration of inhaled nitric oxide preserves alveolar-capillary membrane integrity in porcine gram-negative sepsis; Bloomfield GL et al.; OBJECTIVE: To determine the effect of delayed administration of inhaled nitric oxide (NO) on acute lung injury after the onset of gram-negative sepsis . DESIGN: Nonrandomized controlled study . SETTING: University medical center laboratory . SUBJECTS: Yorkshire swine . INTERVENTIONS: Five groups of swine were anesthetized, mechanically ventilated, and studied for 5 hours . After surgical preparation, control (n = 10) and NO-treated control (n = 6) animals received a 1-hour infusion of sterile saline solution . Sepsis was induced with a 1-hour intravenous infusion of live Pseudomonas aeruginosa . Untreated animals with sepsis (n = 10) received no treatment . Inhaled NO at 20 ppm was administered to NO30-treated animals with sepsis (n = 7) and NO60-treated animals with sepsis (n = 8) beginning at 30 and 60 minutes after bacterial infusion was begun, respectively . MAIN OUTCOME MEASURES: Systemic and pulmonary hemodynamics, arterial blood gas determination, bronchoalveolar lavage protein and neutrophil content, neutrophil oxidant burst, lung myeloperoxidase content, and scanning electron micrographic studies . RESULTS: A progressive, significant (P < .05) decline in PaO2 developed in untreated animals with sepsis, which was prevented in NO30- and NO60-treated animals with sepsis . A significant (P < .05) increase in bronchoalveolar lavage protein and neutrophil counts compared with baseline values was observed in untreated animals with sepsis, indicating acute lung injury . These variables exhibited no notable increase in NO30- and NO60-treated animals with sepsis and were significantly (P < .05) reduced compared with untreated animals with sepsis . The lung myeloperoxidase content was significantly (P < .05) elevated at 5 hours in all groups with sepsis compared with baseline values and the control and NO-treated control groups . The total phorbol myristate acetate-induced polymorphonuclear leukocyte oxidant burst at 5 hours was significantly (P < .05) decreased in the NO30- and NO60-treated animals with sepsis compared with untreated animals with sepsis . Untreated and NO30- and NO60-treated animals with sepsis showed a significant (P < .05) increase in pulmonary artery pressure at 30 minutes, followed by a progressive decline . These changes were significant (P < .05) compared with baseline values and the control groups . No significant (P < .05) difference in pulmonary artery pressure or systemic arterial pressure was found at any time between untreated and NO30- and NO60-treated animals with sepsis . CONCLUSIONS: The delayed administration of inhaled NO preserves alveolar-capillary membrane integrity in this porcine model of gram-negative sepsis . The inhibition of neutrophil transendothelial migration, rather than neutrophil rolling or tight adhesion, may be a critical mechanism by which inhaled NO produces this effect . Decreased oxidant production by activated neutrophils may be a secondary mechanism by which inhaled NO reduces acute lung injury.

Mol Microbiol, 1997 Jan, 23(1), 109 - 21
The Myxococcus xanthus pilT locus is required for social gliding motility although pili are still produced; Wu SS et al.; Social gliding motility in Myxococcus xanthus depends on the presence of Type IV pili . To begin to examine the role of pili in social motility, 17 mutants were identified which had lost social motility, but still expressed pili . Four of these mutants carry point mutations which mapped to a locus upstream of the recently identified pilS, pilR, and pilA genes . Sequencing of this locus revealed a gene with homology to pilT from Pseudomonas Aeruginosa . Sequencing of the four point mutations revealed that they occurred within the M . xanthus pilT locus . A markerless deletion within M . xanthus pilT, similar to the four point mutations, disrupted social gliding behaviour but did not interfere with pilus formation or pilus-dependent cell-cell agglutination . Using time-lapse videomicroscopy, residual social motility was observed in dsp- strains (known to be deficient in fibril but not pilus production); this was not observed in a delta pilT dep- double mutant . Two genes flanking pilT were also sequenced, and found to have homology to pilB and pilC from P . aeruginosa . Markerless deletions within these genes caused both pilus and social-motility defects . These results indicate that M . xanthus pilB and pilC are required for pilus biogenesis, while pilT is required for assembled pili to play their role in social motility . Thus, pilB, pilT, pilC, pilS, pilR and pilA form a contiguous cluster of pil genes required for social motility.

Mol Microbiol, 1997 Jan, 23(1), 43 - 56
Molecular characterization of mutants affected in the osmoprotectant-dependent induction of phospholipase C in Pseudomonas aeruginosa PAO1; Sage AE et al.; Production of the two phospholipases C (PLCs) in Pseudomonas aeruginosa PAO1 is induced under conditions of phosphate limitation, or by the osmoprotectants choline or glycine betaine . Tn5 mutagenesis was performed on strain PAO1 to isolate mutants deficient in choline-dependent induction of PLC . Two mutants, Tn5T1 and Tn5G19, were identified which produce decreased levels of PLC in phosphate-replete media supplemented with choline . A total of 136 and 496 bp of flanking DNA from Tn5G19 and Tn5T1 was cloned by an inverse polymerase chain reaction (PCR) and sequenced . The DNA flanking the Tn5T1 insertion contains an open reading frame predicted to encode a peptide that is approx . 60% identical to the N-terminus of a previously identified protein (P35) of unknown function from Escherichia coli . The P35 gene, which is located in the nusA-infB operon in E . coll, was designated orp (osmoprotectant regulator of PLC) . Haemolytic titres, total PlcH protein and beta-galactosidase activity expressed from a chromosomally inserted plcH-lacZ operon fusion were reduced in strain Tn5T1 in comparison with the parental strain (PAO1) carrying the same fusion . However, this mutant expressed several-fold higher levels of plcH message than strain PAO1 in the presence of choline, while the phosphate-starvation-dependent transcript of plcH could not be detected in this mutant . The defects in Tn5T1 are complemented by a DNA fragment, isolated from a genomic library of PAO1, that carries the orp gene . The deduced amino acid sequence of the DNA fragment cloned from Tn5G19 exhibits 84% identity with the betB gene product of E . coli that has betaine aldehyde dehydrogenase activity . This enzyme catalyses the conversion of betaine aldehyde to glycine-betaine . Unlike the parental strain, the Tn5G19 mutant could not utilize choline as a sole carbon, nitrogen and energy source, and it was deficient in betaine aldehyde dehydrogenase activity . Also, consistent with a disruption of betB in Tn5G19, choline inhibited growth of this strain in media containing 0.7 M NaCl, while glycine-betaine restores growth to wild-type levels . The defects in Tn5G19 are complemented by a DNA fragment from PAO1 that carries the betB gene . The orp gene is located between 0.6 to 6.6 min while betB is located between 10.5 to 12.5 min on the chromosome of PAO1.

Pediatr Infect Dis J, 1997 Jan, 16(1), 112 - 7; discussion 123-6
Pharmacokinetic disposition of sequential intravenous/oral ciprofloxacin in pediatric cystic fibrosis patients with acute pulmonary exacerbation; Rubio TT et al.; OBJECTIVE: Information about the pharmacokinetics of fluoroquinolone antibiotics in high risk children is scant . This study examined the disposition of sequentially administered intravenous and oral ciprofloxacin, as well as provided dosing recommendations, for the treatment of acute pulmonary exacerbations in pediatric cystic fibrosis patients . METHODS: After enrollment in a Food and Drug Administration approved protocol, the pharmacokinetic profiles of ciprofloxacin (CIP) administered to 18 children with cystic fibrosis (ages 5 to 17 years) were studied at steady state after sequentially administered intravenous (10 mg/kg every 8 h) and oral (20 mg/kg every 12 h) doses . All children enrolled met published criteria for exacerbation of Pseudomonas aeruginosa lung infection and received CIP intravenously (given as a 1-h infusion) followed by oral administration, each for a minimum of 3 days . All patients were at a mild to moderate stage in their disease with National Institutes of Health scores between 37 and 83 . Blood samples were drawn at 0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0 (after both i.v . and oral dosing) and 12 h (oral only) after CIP administration . CIP serum concentrations were determined by high performance liquid chromatography . RESULTS: After oral CIP mean +/- SD peak serum concentrations and peak times were 3.7 +/- 1.4 mg/l and 2.5 +/- 1.8 h, respectively, compared with 5.0 +/- 1.5 mg/l and 1.0 +/- 0.3 h after completion of the i.v . infusion . Maximum concentrations, when normalized for dose, were 0.52 +/- 0.12 and 0.19 +/- 0.07 mg/l/kg after i.v . and oral dosing, respectively . The mean bioavailability of oral CIP for all patients was 76%; younger patients appeared to absorb oral CIP less than older subjects, 68% vs . 95%, respectively . For all patients elimination half-lives were 2.6 +/- 0.6 and 3.4 +/- 0.7 h after i.v . and oral administration, respectively, and did not differ by age . Total clearance after i.v . administration was 19.5 +/- 10.9 liters/h . No significant CIP-related adverse effects were noted . CONCLUSIONS: CIP doses of 30 mg/kg/day i.v . and 40 mg/kg/day orally must be administered to children with cystic fibrosis to achieve optimal therapeutic concentrations.

Pediatr Infect Dis J, 1997 Jan, 16(1), 106 - 11; discussion 123-6
Ciprofloxacin as antipseudomonal treatment in patients with cystic fibrosis; Schaad UB et al.; OBJECTIVE: The efficacy and safety of oral ciprofloxacin as a maintenance antipseudomonal therapy were evaluated in 44 patients with cystic fibrosis who had completed a 14-day regimen of intensive hospital therapy with intravenous ceftazidime and amikacin, supplemented by amikacin inhalation therapy . METHODS: Twenty-one patients were randomly assigned to oral ciprofloxacin alone (Group I) and 23 received ciprofloxacin plus inhaled amikacin (Group II) . RESULTS: Negative sputum cultures were achieved in 34 patients (77%) at the end of intensive therapy (19 Group I and 15 Group II) and were sustained after 3 months of maintenance therapy in 5 of the 19 responders in Group I (26%) and in 8 of the 15 responders in Group II (53%) . Resistance to ciprofloxacin was found in 7 of 31 (23%) sputum isolates at the end of ciprofloxacin therapy . During maintenance therapy, continued improvement in clinical symptoms was observed in 14 patients in both treatment groups; 6 in each group had further improvements whereas only 4 patients were clinical failures . There was no correlation between clinical outcome and either elimination of Pseudomonas aeruginosa from sputum culture or development of ciprofloxacin resistance . Both maintenance regimens were well-tolerated by this population of patients which included 28 children younger than 15 years of age . There were no severe or serious adverse events, no signs of quinolone-related arthropathy and no growth impairment . CONCLUSION: Ciprofloxacin was efficacious, safe and well-tolerated as maintenance antipseudomonal therapy in cystic fibrosis patients . These results suggest further evaluation of ciprofloxacin as an oral maintenance therapy is warranted.

Pediatr Infect Dis J, 1997 Jan, 16(1), 97 - 105; discussion 123-6
Sequential ciprofloxacin therapy in pediatric cystic fibrosis: comparative study vs . ceftazidime/tobramycin in the treatment of acute pulmonary exacerbations . The Cystic Fibrosis Study Group; Church DA et al.; BACKGROUND: Cystic fibrosis patients have chronic bacterial infections of the respiratory tract, most commonly Pseudomonas aeruginosa . Although controversial, administration of antibiotic therapy during acute pulmonary exacerbations is standard practice . Fluoroquinolones are currently not indicated for use in young children because of the observation of arthropathy and damage to growing cartilage in beagle puppies . Because of its activity against P . aeruginosa and excellent oral bioavailability, ciprofloxacin offers a unique therapeutic alternative for this patient population . OBJECTIVE: This prospective, randomized, double blind study compared the efficacy and safety of sequential intravenous/oral ciprofloxacin vs . ceftazidime/tobramycin in hospitalized pediatric cystic fibrosis patients with an acute pulmonary exacerbation associated with P . aeruginosa infection . METHODS: One hundred thirty patients (ages 5 to 17 years) were randomized to receive either i.v . ciprofloxacin 10 mg/kg every 8 h for 7 days followed by oral ciprofloxacin 20 mg/kg every 12 h for a minimum of 3 days or i.v . ceftazidime 50 mg/kg every 8 h plus i.v . tobramycin 3 mg/kg every 8 h for a minimum of 10 days . Clinical, bacteriologic and safety responses were assessed throughout the study . RESULTS: All 84 patients (median age, 11 years; range, 5 to 17 years) valid for efficacy in both treatment groups demonstrated clinical improvement . Five patients experienced clinical relapses (3 ciprofloxacin, 2 ceftazidime/tobramycin) by the 2- to 4-week follow-up . Intent-to-treat analysis demonstrated similar clinical findings between the two treatment groups at both the end of therapy and follow-up . Clinical improvement correlated with improvement in pulmonary function studies and the acute clinical scoring system but not with bacteriologic eradication of Pseudomonas . DNA profiles demonstrated that irrespective of colony morphology, usually one clonal strain was associated with each patient's pulmonary exacerbation . Treatment-associated musculoskeletal events occurred with equal frequency (22% vs . 21%) in both study drug groups (n = 129), and arthralgias were within the range of rates for cystic fibrosis arthropathy . None of these events required study drug discontinuation . CONCLUSION: Sequential i.v./oral ciprofloxacin monotherapy offers a safe and efficacious alternative to standard parenteral therapy for acute pulmonary exacerbations in pediatric cystic fibrosis patients.

CLAO J, 1997 Jan, 23(1), 63 - 8
Adherence of Pseudomonas aeruginosa to shed rabbit corneal epithelial cells after overnight wear of contact lenses; Ren H et al.; PURPOSE: Previous studies have shown that contact lens oxygen transmissibility correlates with binding of Pseudomonas aeruginosa to the rabbit cornea after overnight lens wear . Studies of human lens wear stratified by oxygen transmissibility will be required to validate these animal results . In humans, bacterial binding to shed cells obtained through corneal irrigation cytology may provide an indirect measure of in vitro binding . The purpose of this study was to establish the relationship between binding to shed cells and to the residual corneal surface in an animal model of lens wear prior to initiation of human studies . METHODS: The test contact lenses used were: rigid lens A (Dk/L = 10 x 10(-9) {cm/ sec}{mL O2/mL mmHg}); rigid lens B (Dk/L = 97); soft lens A (Dk/L = 9); soft lens B (Dk/L = 20); and, soft lens C (Dk/L = 39) . There were six rabbits in each group, except for the soft lens C group, which had seven rabbits . After overnight lens wear, the corneal surface was irrigated with a corneal irrigation chamber to collect surface cells before exposure to a bacterial suspension (1 x 10(7) CFU/mL) for 30 minutes . The number of bacteria adherent to the residual corneal surface was then assessed by CFU determination . Cells collected from the corneal surface (9 mL) were incubated with 1 mL bacterial suspension containing 10(8) (CFU/mL) for 30 minutes . The number of bacteria adherent to shed cells was assessed by staining with acridine orange and direct counting by epifluorescence microscopy . RESULTS: The differences in the number of bacteria adhering to shed epithelial cells between the treated and the control eyes were 2.90 +/- 1.20 and 0.23 +/- 0.41 for rigid lenses A and B, respectively, and 5.97 +/- 1.54, 3.67 +/- 2.32, and 0.90 +/- 1.45 (bacterial/cell) for soft lenses A, B, and C, respectively . Overnight contact lens wear induced a significant increase in bacterial binding to shed corneal epithelial cells for rigid lens A and for soft lenses A and B . There were significant differences among lens groups (P = 0.00017, ANOVA), with significant differences between rigid lenses A and B, soft lenses A and C, and soft lenses B and C . The binding of bacteria to shed cells was significantly correlated with the binding of bacteria to the residual corneal surface, both confirming and extending previous results (R = 0.78, P < 0.001) . CONCLUSION: These results demonstrate a positive correlation between P . aeruginosa adherence to shed corneal cells and to the residual corneal surface in the rabbit eye following contact lens wear . In light of the results from prior animal studies, examination of the behavior of P . aeruginosa binding to exfoliated cells appears to be a promising and valid method for future assessment of similar lens-induced increases in bacterial binding in prospective human clinical studies.

CLAO J, 1997 Jan, 23(1), 57 - 62
Loss of bactericidal activity from contact lens storage solutions; Rosenthal RA et al.; PURPOSE: The purpose of this study was to evaluate the effect of lens storage on the bactericidal activities of disinfecting solutions . METHODS: HYDROCURVEII (55% water content) soft contact lenses were soaked in OPTI-FREE Rinsing, Disinfecting, and Storage Solution (preserved with polyquaternium-1 {PQ-1}) or ReNu Multi-Purpose Solution (polyaminopropyl biguanide {PHMB}) . After the lenses were removed, solutions were challenged with Pseudomonas aeruginosa and Staphylococcus aureus . Uptake of the antimicrobial agents by the lenses was evaluated by placing the lenses on plates seeded with S . aureus and observing for zones of growth inhibition . RESULTS: The bactericidal activity of the PHMB preserved solution decreased after 4 hours of storage time and showed no activity after storage for 3 days . Lenses soaked in the PHMB preserved solution produced zones of inhibition . The results suggest that the PHMB in the solution accumulates in the lens . This decreases the levels of preservative in the solution that is no longer available during lens storage . Neither decrease in bactericidal activity nor accumulation of the PQ-1 preservative in the lens occurred during storage with the PQ-1 solution . CONCLUSION: These results demonstrate that PQ-1 preserved solutions maintain bactericidal activity overnight and during prolonged storage without detectable uptake by lenses.

Am J Respir Crit Care Med, 1997 Jan, 155(1), 337 - 42
Effect of clarithromycin on lymphocytes in chronic respiratory Pseudomonas aeruginosa infection; Yanagihara K et al.; In a newly established murine model of chronic Pseudomonas aeruginosa respiratory infection mimicking diffuse panbronchiolitis (DPB), we investigated the effect of oral administration of clarithromycin on lymphocyte accumulation in the lung . Infection was produced by placement of a plastic tube precoated with P . aeruginosa in the bronchus . The number of bacteria on the tube was 6.25 +/- 0.22 log10 colony-forming units (cfu)/ml . Viable bacteria were constantly isolated at 10(5) to 10(6) cfu/specimen from the lungs for more than 1 yr . The histopathologic features resembled those of DPB consisting of massive accumulation of lymphocytes in the lung . The total number of pulmonary lymphocytes started to increase on Day 7, reaching a peak level within 12 d of intratracheal challenge . The number remained steady at that level for up to 120 d . There was also a steady fall in the CD4+/CD8+ ratio in the lungs, commencing on Day 7 and persisting to Day 120 . A 10-d course of oral clarithromycin (10 mg/kg/d) from Day 7 resulted in a reduction of lymphocyte numbers to baseline level, although the dose did not influence the number of bacteria in the lungs . Treatment also increased the CD4+/CD8+ ratio to the baseline level from Day 7 to 17 . Our results were similar to those detected in bronchoalveolar lavage fluid of patients with DPB and suggest that the therapeutic benefits of clarithromycin are due to its anti-inflammatory properties rather than antimicrobial effect.

Am J Respir Crit Care Med, 1997 Jan, 155(1), 327 - 36
Effect of salmeterol on Pseudomonas aeruginosa infection of respiratory mucosa; Dowling RB et al.; We have studied the effect of salmeterol on both P . aeruginosa interactions with the mucosa of nasal turbinate organ cultures and on pyocyanin-induced (20 microg/ml) and elastase-induced (100 microg/ml) damage to nasal epithelial cells . Organ cultures were exposed to salmeterol either by preincubation with 4 x 10(-7) M salmeterol for 30 min or by pipetting 20 microl of 4 x 10(-7) M salmeterol onto the organ culture surface immediately prior to bacterial inoculation . Infected organ cultures (8 h) had significantly (p < or = 0.01) increased epithelial damage, and P . aeruginosa was predominantly associated with damaged epithelium and mucus . Salmeterol significantly (p < or = 0.02) reduced epithelial damage caused by infection and the total number of adherent bacteria (p < or = 0.05), but bacterial distribution on the mucosa was unchanged . Nasal epithelial cells incubated with pyocyanin (20 microg/ml) or elastase (100 microg/ml) for 3 h had significantly (p < or = 0.05) increased cytoplasmic blebbing and mitochondrial damage versus control values . Elastase also significantly (p < or = 0.05) increased cell projection and reduced the level of ciliation . Cells preincubated with salmeterol (2 x 10(-7) M) showed a significant reduction in some features of cell damage caused by both toxins, which was inhibited by the beta2-adrenoceptor antagonist propranolol . Our results indicate that salmeterol reduces P . aeruginosa-induced damage to both organ culture and nasal epithelium.

Arch Microbiol, 1997 Jan, 167(1), 6 - 10
Respiratory stimulation and generation of superoxide radicals in Pseudomonas aeruginosa by fungal naphthoquinones; Haraguchi H et al.; The mechanism of action of antimicrobial naphthoquinones from the fungus Fusarium was studied by using Pseudomonas aeruginosa . Bostricoidin, methyl ether fusarubin, and fusarubin stimulated the oxygen consumption of bacterial cells and induced cyanide-insensitive oxygen consumption . These activities of the tested compounds were also observed in bacterial membrane preparations in a dose-dependent manner . Naphthoquinones stimulated the generation of superoxide anion and hydrogen peroxide . The naphthoquinone effectively acted as the electron acceptors for bacterial diaphorase, which could explain the antibacterial activity of Fusarium naphthoquinones since electron acceptors lead to the stimulation of respiratory activity and the generation of oxygen radical species.

Chemotherapy, 1997 Jan-Feb, 43(1), 27 - 30
Susceptibility of Pseudomonas aeruginosa isolates to ceftazidime is unrelated to the expression of the outer membrane protein OprC; Perez FJ et al.; Previously, it has been postulated that the porin OprC facilitates the diffusion of ceftazidime through the outer membrane of Pseudomonas aeruginosa . To further investigate this claim, the outer membrane protein (OMP) profiles of 22 ceftazidime-susceptible clinical isolates were analyzed . No correlation was found between MIC values and the level of expression of OprC . Further, OprC was either undetectable or expressed in reduced amounts in 12 isolates . In contrast, OprF and OprE were present in all isolates studied . This study suggests that OprC is dispensable for the permeation of ceftazidime through the outer membrane of P . aeruginosa.

Cornea, 1997 Jan, 16(1), 101 - 6
Effect of hybrid peptides of cecropin A and melittin in an experimental model of bacterial keratitis; Nos-Barbera S et al.; Synthetic peptides, ranging from 12 to 18 residues, containing partial sequences from natural cecropin A and melittin were tested for activity in an experimental pseudomonas keratitis model in rabbits . In separate experiments, two Pseudomonas aeruginosa strains: (a) a clinical isolated strain, and (b) an American Type Culture Collection (ATCC) strain, were inoculated into the stroma of one cornea of each rabbit . Peptides were topically applied at 0.1% in phosphate-buffered saline (PBS) and compared with PBS alone and 0.3% gentamicin eye drops . Clinical evaluation, based on the McDonald-Shadduck scale, was performed during a > 48-h period after the bacterial inoculation . The peptide-treated animals showed significantly lower (p < 0.05) inflammatory signs and lower anterior-segment bacterial damage compared with PBS-treated animals, after the first 6 h . The antiinflammatory/antimicrobial activity was non significantly differnt (p > 0.05) from that in animals treated with gentamicin . We conclude that peptides keeping the sequence KWKLFKK from cecropin A and at least the sequence VLKVL from melittin show promise as novel agents in topical ocular therapy of bacterial keratitis.

J Bacteriol, 1997 Jan, 179(1), 248 - 57
Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa; Serino L et al.; The high-affinity siderophore salicylate is an intermediate in the biosynthetic pathway of pyochelin, another siderophore and chelator of transition metal ions, in Pseudomonas aeruginosa . The 2.5-kb region upstream of the salicylate biosynthetic genes pchBA was sequenced and found to contain two additional, contiguous genes, pchD and pchC, having the same orientation . The deduced amino acid sequence of the 60-kDa PchD protein was similar to those of the EntE protein (2,3-dihydroxybenzoate-AMP ligase) of Escherichia coli and other adenylate-forming enzymes, suggesting that salicylate might be adenylated at the carboxyl group by PchD . The 28-kDa PchC protein showed similarities to thioesterases of prokaryotic and eukaryotic origin and might participate in the release of the product(s) formed from activated salicylate . One potential product, dihydroaeruginoate (Dha), was identified in culture supernatants of iron-limited P . aeruginosa cells . The antifungal antibiotic Dha is thought to arise from the reaction of salicylate with cysteine, followed by cyclization of cysteine . Inactivation of the chromosomal pchD gene by insertion of the transcription and translation stop element omega Sm/Sp abolished the production of Dha and pyochelin, implying that PchD-mediated activation of salicylate may be a common first step in the synthesis of both metabolites . Furthermore, the pchD::omega Sm/Sp mutation had a strong polar effect on the expression of the pchBA genes, i.e., on salicylate synthesis, indicating that the pchDCBA genes constitute a transcriptional unit . A full-length pchDCBA transcript of ca . 4.4 kb could be detected in iron-deprived, growing cells of P . aeruginosa . Transcription of pchD started at tandemly arranged promoters, which overlapped with two Fur boxes (binding sites for the ferric uptake regulator) and the promoter of the divergently transcribed pchR gene encoding an activator of pyochelin biosynthesis . This promoter arrangement allows tight iron-mediated repression of the pchDCBA operon.

J Bacteriol, 1997 Jan, 179(1), 235 - 42
Gene cluster for dissimilatory nitrite reductase (nir) from Pseudomonas aeruginosa: sequencing and identification of a locus for heme d1 biosynthesis; Kawasaki S et al.; The primary structure of an nir gene cluster necessary for production of active dissimilatory nitrite reductase was determined from Pseudomonas aeruginosa . Seven open reading frames, designated nirDLGHJEN, were identified downstream of the previously reported nirSMCF genes . From nirS through nirN, the stop codon of one gene and the start codon of the next gene were closely linked, suggesting that nirSMCFDLGHJEN are expressed from a promoter which regulates the transcription of nirSM . The amino acid sequences deduced from the nirDLGH genes were homologous to each other . A gene, designated nirJ, which encodes a protein of 387 amino acids, showed partial identity with each of the nirDLGH genes . The nirE gene encodes a protein of 279 amino acids homologous to S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from other bacterial strains . In addition, NirE shows 21.0% identity with NirF in the N-terminal 100-amino-acid residues . A gene, designated nirN, encodes a protein of 493 amino acids with a conserved binding motif for heme c (CXXCH) and a typical N-terminal signal sequence for membrane translocation . The derived NirN protein shows 23.9% identity with nitrite reductase (NirS) . Insertional mutation and complementation analyses showed that all of the nirFDLGHJE genes were necessary for the biosynthesis of heme d1.

J Bacteriol, 1997 Jan, 179(1), 187 - 93
Identification of the algZ gene upstream of the response regulator algR and its participation in control of alginate production in Pseudomonas aeruginosa; Yu H et al.; Alginate production in mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients is under direct control by AlgU, the P . aeruginosa equivalent of the extreme heat shock sigma factor sigma(E) in gram-negative bacteria, and AlgR, a response regulator from the superfamily of two-component signal transduction systems . In this report, we describe the identification of the algZ gene, located immediately upstream of algR, which is involved in the control of alginate production . The predicted product of the algZ gene showed similarity to a subset of sensory components from the superfamily of signal transduction systems but lacked several of the highly conserved motifs typical of histidine protein kinases . Inactivation of algZ in the wild-type standard genetic strain PAO1 did not affect its nonmucoid morphology . However, inactivation of algZ in a mucoid mutant P . aeruginosa strain, which had AlgU freed from control by the anti-sigma factor MucA, resulted in increased alginate production under growth conditions which did not permit expression of mucoidy in the parental algZ+ strain . The observed effects were abrogated when algR was inactivated in the algZ::Tc(r) background . These findings indicate that algZ plays a regulatory role in alginate production, possibly interacting with AlgR, and that it may have negative effects on expression of the mucoid phenotype under the conditions tested . The presented results suggest that elements of negative regulation exist at the levels of both the alternative sigma factor AlgU and the transcriptional activator AlgR which, once relieved from that suppression, cooperate to bring about the expression of the alginate system.

J Bacteriol, 1997 Jan, 179(1), 17 - 23
The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant; Heuner K et al.; Gene expression in Legionella pneumophila, the etiological agent of Legionnaires' disease, can be controlled by alternative forms of RNA polymerase programmed by distinct sigma factors . To understand the regulation of L . pneumophila flagellin expression, we cloned the sigma factor (FliA) of RNA polymerase responsible for the transcription of the flagellin gene, flaA . FliA is a member of the sigma28 class of alternative sigma factors identified in several bacterial genera . The gene fliA has been isolated from an expression library of L . pneumophila isolate Corby in Escherichia coli K-12 . This library was transformed into a fliA mutant of E . coli K-12 containing a plasmid carrying the L . pneumophila-specific flaA promoter fused to the reporter gene luxAB . Screening the obtained transformants for luciferase activity, we isolated the major part of the fliA gene on a 1.64-kb fragment . This fragment was sequenced and used for reverse PCR in order to recover the complete fliA gene . The resulting 1.03-kb fragment was shown to contain the entire fliA gene . L . pneumophila FliA has 55 and 43% amino acid identity with the homologous sequences of Pseudomonas aeruginosa and E . coli . Furthermore, the L . pneumophila fliA gene was able to restore the flagellation and the motility defect of an E . coli fliA mutant . This result suggests that the L . pneumophila sigma28 protein can bind to the E . coli core RNA polymerase to direct transcription initiation from the flaA-specific promoter.

Antimicrob Agents Chemother, 1997 Jan, 41(1), 95 - 100
Synergism between tobramycin and ceftazidime against a resistant Pseudomonas aeruginosa strain, tested in an in vitro pharmacokinetic model; den Hollander JG et al.; Synergism between two antibiotics is usually tested by a checkerboard titration technique, or by time-kill methods . Both methods have the disadvantage that synergism is determined at constant concentrations of the antibiotics, which do not reflect reality in vivo . In the present study we determined whether synergism between tobramycin and ceftazidime can be found at declining concentrations below the MIC, and whether change in dosing sequence of the antibiotics would result in differences in killing . Three monotherapy and six combination therapy schedules were tested in an in vitro pharmacokinetic model, using a Pseudomonas aeruginosa resistant to both antibiotics . During all q8h dosing schedules the peak concentration (Cmax) was adjusted to the MIC for the strain of both antibiotics . During all monotherapy regimens bacterial growth was present, while all six combination therapy schedules showed significant killing . At t = 24 h there were no differences between all combination therapy schedules, but at t = 8 h the two combination therapy schedules with administration of tobramycin once daily showed a significantly faster killing . By using the area under the killing curve (AUKC) as a parameter for synergistic killing, simultaneous combination therapy starting with tobramycin once daily was significantly better than all other regimens . We conclude that there is synergism between tobramycin and ceftazidime at declining antibiotic concentrations below the MIC, resulting in a pronounced killing of a resistant Pseudomonas strain . Infections due to resistant Pseudomonas strains could possibly be treated by a synergistic combination of these drugs.

Infect Immun, 1997 Jan, 65(1), 248 - 56
Effects of differential expression of the 49-kilodalton exoenzyme S by Pseudomonas aeruginosa on cultured eukaryotic cells; Olson JC et al.; Production of the ADP-ribosylating enzyme exoenzyme S (ExoS) by Pseudomonas aeruginosa has been associated with increased virulence . Previous studies, however, have been unable to confirm an effect of soluble ExoS in cell culture or animal model systems . To determine if bacteria must come in contact with target cells in order for an effect of ExoS to be observed, coculture systems were developed to compare the effects of ExoS- and non-ExoS-producing bacteria on eukaryotic cell function . The two P . aeruginosa strains used in these studies, 388 and 388delta exoS, maintained genetic identity, with the exception that strain 388delta exoS lacked production of the 49-kDa form of ExoS . When bacteria were cocultured with Detroit 532 fibroblastic cells, ExoS-producing 388 bacteria caused a significant decrease in DNA synthesis and viability compared to the decrease caused by non-ExoS-producing 388delta exoS bacteria . Maximal differences between the two strains were observed when 10(4) to 10(7) CFU of bacteria/ml were cocultured with Detroit cells for 4 or 6 h . Both strains were effective in eliminating Detroit cell DNA synthesis after a 20-h coculture period . Secreted ExoS had no effect on Detroit cell growth and viability, indicating that bacteria must have contact with target cells for the effect of ExoS on cellular function to be observed . Similar effects on cell proliferation and viability were observed when the two strains were cocultured with the KB epithelioid cell line . ExoS-associated decreases in eukaryotic cell viability were not found to be mediated by an inhibition of protein synthesis . These studies confirm that the 49-kDa ExoS contributes to the cellular pathogenesis of P . aeruginosa by interfering with eukaryotic cell growth and viability . In addition, the coculture system developed which recognizes this effect should provide a means for defining the function of ExoS in vivo.

Infect Immun, 1997 Jan, 65(1), 89 - 94
Induction of tumor necrosis factor production from monocytes stimulated with mannuronic acid polymers and involvement of lipopolysaccharide-binding protein, CD14, and bactericidal/permeability-increasing factor; Jahr TG et al.; Well-defined polysaccharides, such as beta1-4-linked D-mannuronic acid (poly{M}) derived from Pseudomonas aeruginosa, induce monocytes to produce tumor necrosis factor (TNF) through a pathway involving membrane CD14 . In this study we have investigated the effects of soluble CD14 (sCD14), lipopolysaccharide-binding protein (LBP), and bactericidal/permeability-increasing factor (BPI) on poly(M) binding to monocytes and induction of TNF production . We show that LBP increased the TNF production from monocytes stimulated with poly(M) . Addition of sCD14 alone had only minor effects, but when it was added together with LBP, a rise in TNF production was seen . BPI was found to inhibit TNF production from monocytes stimulated with poly(M) in the presence of LBP, LBP-sCD14, or 10% human serum . Binding studies showed that poly(M) bound to LBP- and BPI-coated immunowells, while no significant binding of poly(M) to sCD14-coated wells in the absence of serum was observed . Binding of poly(M) to monocytes was also examined by flow cytometry, and it was shown that the addition of LBP or 10% human serum clearly increased the binding of poly(M) to monocytes . BPI inhibited the binding of poly(M) to monocytes in the presence of LBP, LBP-sCD14, or 10% human serum . Our data demonstrate a role for LBP, LBP-sCD14, and BPI in modulating TNF responses of defined polysaccharides.

Biochemistry, 1996 Dec 17, 35(50), 16391 - 8
Effect of pH and ligand binding on the structure of the Cu site of the Met121Glu mutant of azurin from Pseudomonas aeruginosa; Strange RW et al.; A pH-dependent X-ray absorption fine structure (XAFS) study has been undertaken to provide a structural interpretation of the spectroscopic properties of the Met121 Glu mutant of azurin from Pseudomonas aeruginosa (Azp) . Ligand binding studies have been carried out to investigate the effect of the cavity formed at the Cu site as a result of the mutation . The optical spectrum at pH 4 exhibits an intense band at approximately 600 nm and a weaker band at approximately 450 nm, typical for the blue copper proteins . As the pH is increased, these bands decrease in intensity and shift to 570 and 413 nm, respectively, with the latter becoming the more intense of the two {Karlsson, B.G., et al . (1991) Protein Eng . 4 (3), 343-349} . These changes are accompanied by a change in the EPR spectrum from a rhombic type 1 Cu spectrum at pH 4 to a spectrum with the rhombic splitting decreasing to zero and the hyperfine coupling increasing from 25 to 83 G . X-ray absorption a the Cu K-edge shows that this change results from the lengthening of the Cu-His (by 0.07 A) and Cu-Cys (by 0.06 A) bonds and the coordination of one of the oxygen atoms of the glutamate ligand at pH 8, at a distance as close as 1.90 A . The copper site thus changes from a normal type 1 copper center with three strong bonds at pH 4 to a copper site with four strong bonds at pH 8, with Cu-His distances significantly longer than known distances for type 1 copper centres measured using the XAFS technique . The XAFS of the azide derivative measured at pH 8 shows a similar Cu coordination, with azide replacing glutamate as the fourth ligand . Azide binding at pH 8 is accompanied by a further increase in the EPR hyperfine coupling to 110 G . This structural information when taken together with recent structural sudies on copper proteins points toward the need for a reexamination of the basis on which copper proteins are classified.

Gene, 1996 Dec 5, 182(1-2), 137 - 43
Isolation, sequencing and overproduction of the single-stranded DNA binding protein from Pseudomonas aeruginosa PAO; Genschel J et al.; The gene (ssb) encoding the single-stranded DNA binding (SSB) protein from Pseudomonas aeruginosa PAO was detected on a 2.1 kbp PstI-fragment of chromosomal DNA . The protein (PaeSSB) encoded by this gene consists of 165 aa and has a M(r) of 18549 . The genomic sequence was confirmed by amino acid sequencing of the amino terminus of SSB protein isolated from P . aeruginosa PAO . PaeSSB shows 68% homology to the respective protein of E . coli . The nucleotide sequence upstream of the P . aeruginosa ssb gene shows little homology to the regulatory region upstream of the ssb gene of E . coli . The ssb gene was located at a distance of 690-870 kbp from the origin of replication on a physical map of P . aeruginosa PAO . In vivo PaeSSB could replace the SSB protein of E . coli (EcoSSB) if its production was controlled by the lac promoter on a high-copy vector . PaeSSB was overproduced in E . coli . Both the overproduced protein and PaeSSB isolated from Pseudomonas aeruginosa PAO are post-translationally modified by cleavage of the first methionine . Analytical ultracentrifugation shows that PaeSSB is a stable homotetramer . The copy number of PaeSSB in P . aeruginosa is 1200 +/- 250 tetramers per cell . Preliminary characterization of the DNA binding properties shows PaeSSB to have a lower affinity for single-stranded DNA than EcoSSB.

Gene, 1996 Dec 5, 182(1-2), 63 - 70
Molecular analysis of the Pseudomonas aeruginosa genes, ruvA, ruvB and ruvC, involved in processing of homologous recombination intermediates; Hishida T et al.; In Escherichia coli, the products of the ruvA, ruvB and ruvC genes are all involved in the processing of recombination intermediates (Holliday structures) into recombinant molecules . We cloned a 9.4-kb DNA fragment from Pscudomonas aeruginosa PAO1 in a plasmid by functional complementation of the UV sensitivity of an E . coli strain with ruvABC deleted . In P . aeruginosa, the ruv region seemed to form a non-SOS regulated single operon consisting of orf26-ruvC-ruvA-ruvB, while in this region of E . coli, ruvA and ruvB form an SOS-regulated operon, orf26 and ruvC form a non-SOS operon, and these two operons are split by orf23 . The deduced amino acid sequences of P . aeruginosa RuvA, RuvB and RuvC proteins were 55, 72 and 55% identical to those of the corresponding E . coli Ruv proteins . The individual ruv genes of P . aeruginosa complemented the corresponding single ruv mutations of E . coli, suggesting that the P . aeruginosa Ruv proteins can interact functionally with their E . coli Ruv partners in forming heterologous complexes . The sequence alignments of the Ruv proteins were extended by incorporation of data about the putative ruv genes obtained from data banks, and the RuvB sequences were conspicuously more conserved than the RuvA and RuvC sequences.

Biochemistry, 1996 Dec 3, 35(48), 15134 - 42
Investigation into the catalytic role for the tryptophan residues within domain III of Pseudomonas aeruginosa exotoxin A; Beattie BK et al.; The role of the tryptophan residues in the substrate-binding and catalytic mechanism of an enzymatically active C-terminal fragment of Pseudomonas aeruginosa exotoxin A was studied by individually or jointly replacing these residues with phenylalanine . Substitution of W-466 decreased the ADP-ribosyltransferase and NAD(+)-glycohydrolase activities by 20- and 3-fold, respectively . In contrast, substitution of W-417 or W-558 with phenylalanine both resulted in a 3-fold decrease in ADP-ribosyltransferase activity with, however, only a decrease by 40% and 70% in NAD(+)-glycohydrolase activity, respectively . Simultaneous replacement of W-466 and W-558 resulted in a 200-fold decrease in ADP-ribosyltransferase and an 6-fold decrease in NAD(+)-glycohydrolase activities, suggesting that W-466 may play a minor role in the transfer of ADP-ribose to the eEF-2 protein . Chemical modification of the tryptophan residues in the wild-type toxin fragment by N-bromosuccinimide revealed the presence of a single residue important for enzymatic activity, W-466, with a minor contribution from W-558 . Additionally, tryptophan residues, W-305 and W-417, were refractory to oxidation by N-bromosuccinimide, which likely indicated the buried nature of these residues within the protein structure . Titration of the wild-type toxin fragment with NAD+ resulted in the quenching of the intrinsic tryptophan fluorescence to 58% of the initial value . Titration of the various single and a double tryptophan replacement mutant protein(s) indicated that W-558 and W-466 are responsible for the substrate-induced fluorescence quenching, with the former being responsible for the largest fraction of the observed quenching in the wild-type toxin . Consequently, a molecular mechanism is proposed for the substrate-induced fluorescence quenching of both W-466 and W-558 . Furthermore, molecular modeling of the recent crystal structures for both exotoxin A (domain III fragment) and diphtheria toxin, combined with a variety of previous results, has led to the proposal for a catalytic mechanism for the ADP-ribosyltransferase reaction . This mechanism features a SN1 attack (instead of the previously purported SN2 mechanism) by the diphthamide residue (nucleophile) of eukaryotic elongation factor 2 on the C-1 of the nicotinamide ribose of NAD+, which results in an inversion of configuration likely due to steric constraints within the NAD(+)-toxin-elongation factor 2 complex.

Br J Radiol, 1996 Dec, 69(828), 1099 - 103
Pseudomonas aeruginosa bronchopulmonary infection in patients with advanced human immunodeficiency virus disease; Traill ZC et al.; Pseudomonas aeruginosa is increasingly reported as a respiratory pathogen in patients with advanced human immunodeficiency virus (HIV) disease . We retrospectively reviewed the chest radiographic appearances of 29 HIV-infected adults with bronchopulmonary infection in whom Pseudomonas aeruginosa was the sole respiratory pathogen isolated . The commonest radiographic abnormality was a diffuse reticular (11 patients) or reticulonodular (9 patients) infiltrate in the pulmonary interstitium . Alveolar opacification was seen in seven patients . Cavitation was rare (2 patients), as was ground-glass opacification (2 patients) . Five patients had pleural effusions . No patient had mediastinal or hilar lymphadenopathy . Normal chest radiographs were seen in eight patients . Although the radiographic appearances of Pseudomonas bronchopulmonary infection in HIV-infected patients are non-specific, an interstitial infiltrate is a common finding . Pseudomonas aeruginosa should be considered along with the commoner pathogen Pneumocystis carinii in the differential diagnosis of an interstitial infiltrate in this group of patients.

Antimicrob Agents Chemother, 1996 Dec, 40(12), 2894 - 7
In vivo oral efficacy of levofloxacin for treatment of systemic Pseudomonas aeruginosa infections in a murine model of septicemia; Yagel SK et al.; The in vivo efficacies of levofloxacin and ciprofloxacin in lethal, systemic Pseudomonas aeruginosa infections in mice were compared . MICs of levofloxacin and ciprofloxacin ranged from 0.5 to 2.0 micrograms/ml and from 0.12 to 1.0 microgram/ml respectively . Infecting doses ranged from 5.0 x 10(1) to 3.2 x 10(3) CFU per mouse, depending on the isolate . Test fluoroquinolones were administered orally at 1 h (single dose) or at 1 and 3 h (divided dose) postinfection, with 10 infected mice used for each of six concentrations of each fluoroquinolone tested (1 to 40 mg/kg of body weight) in each dosing regimen . Whether given in a single or a divided dose, the total daily dose was the same for each fluoroquinolone . For mice treated 1 h postinfection with levofloxacin and ciprofloxacin, the effective doses for 50% of the infected mice ranged from 2.09 to 13.80 mg/kg and from 2.34 to 11.22 mg/kg, respectively, and for those treated 1 and 3 h postinfection, the effective doses for 50% of the infected mice ranged from 3.71 to 16.98 mg/kg and from 2.95 to 13.18 mg/kg, respectively . Although the potency varied for both levofloxacin and ciprofloxacin among all strains of P . aeruginosa tested, there were small differences within the same strain for levofloxacin and ciprofloxacin when given in the same dosing regimen . Levofloxacin proved nearly as effective as ciprofloxacin against a systemic P . aeruginosa infection in mice.

Rhinology, 1996 Dec, 34(4), 232 - 6
Sinus infection in intensive care patients; Mevio E et al.; Sinusitis is a complication known to accompany nasotracheal intubation, but its frequency has not been well established . During a two-year-period, 1,126 patients in an intensive care unit have been studied . Twenty-seven of them (2%) developed a bacterial sinusitis . The diagnosis is established on the basis of an unexplained clinical sepsis, imaging evidence of fluid in the maxillary sinus, and antral puncture . Microbiological samples showed Gram-negative micro-organisms, in particular Pseudomonas aeruginosa, and an elevated percentage of Staphylococcus aureus and Escherichia coli . The likely predisposing factors (nasogastric and/or nasotracheal tubes) are discussed . Aetiology, diagnosis and management of the disease are discussed in detail . The importance of prompt removal of nasal instrumentation and of early sinus drainage, in addition to broad-spectrum antibiotic therapy, is emphasized.

Rhinology, 1996 Dec, 34(4), 194 - 7
Is nasal polyposis in cystic fibrosis a direct manifestation of genetic mutation or a complication of chronic infection?
De Gaudemar I, Contencin P, Van den Abbeele T, Munck A, Navarro J, Narcy P.
Cystic fibrosis (CF) is the most common autosomal recessive disease among Caucasians . It is characterized by abnormal transepithelial sodium and chloride transport . The clinical expressions of the disorder are highly variable including nasal polyposis . Some authors have found that CF children with nasal polyposis form a distinct subgroup of patients within the clinical heterogeneity of the disease with milder gastrointestinal and pulmonary symptoms . The aim of this prospective study was to verify whether the clinical manifestations in CF children with nasal polyposis are different from control CF patients, and to identify any correlation between a phenotype of nasal polyposis and a genotype . Sixty-six CF children, aged 1-25 years, consecutively underwent ENT examination including nasal endoscopy . Twenty-one had nasal polyposis . The remainder formed the control group . There was no statistical difference in the mode and age of presentation of the disease between the two groups . The clinical manifestations (Schwachman and Kulczycki score, colonization by Staphylococcus aureus and Pseudomonas aeruginosa) were comparable between the two groups . We found no statistical difference in the repartition of genotypes between the polyposis and the control groups . Nasal polyposis does not seem to be genetically dependent, but a larger sample of patients is needed to reach an accurate conclusion.

Arch Pediatr, 1996 Dec, 3(12), 1248 - 52
{Liver transplantation in an adolescent with cystic fibrosis}; Thevenot P et al.; BACKGROUND: Orthotopic liver transplantation (OLT) is an effective treatment for patients with cystic fibrosis end stage liver disease, especially those with only mild pulmonary involvement . Long-term follow-up in such transplanted patients is still lacking . CASE REPORT: A 15-year-old girl with cystic fibrosis received an OLT because of severe decompensated cirrhosis . She had been colonized by Pseudomonas aeruginosa for 3 years and had pancreatic insufficiency; she also had mild glucose intolerance . Postoperatively she developed diabetes mellitus requiring insulin therapy for 9 months . Oral cyclosporin was poorly absorbed so that she was given a new emulsion of cyclosporin (Neoral) that was better absorbed . A rapid pubertal catch-up was obtained but the patient remained colonized by Pseudomonas aeruginosa . CONCLUSION: This 3-year postoperative follow-up confirms that OLT can represent a good alternative in those patients with severe liver disease and mild pulmonary involvement.

Intern Med, 1996 Dec, 35(12), 979 - 83
Two cases of severe bronchiectasis successfully treated with a prolonged course of trimethoprim/sulfamethoxazole; Honda T et al.; Two patients with severe bronchiectasis, one patient without other disease and the other with hyper IgE syndrome, were successfully treated with long-term therapy with low doses of trimethoprim and sulfamethoxazole (TMP-SMZ) . Recurrent respiratory infections with productive cough and high fever were resistant to various antibiotics and often disturbed the patients' activities in daily life . However, they showed marked improvement following TMP-SMZ therapy, which was started for methicillin-resistant Staphylococcus aureus (MRSA) infection . MRSA disappeared some months later, but Pseudomonas aeruginosa appeared again in the sputum . Both patients, however, have remained free from symptoms for over one year.

Virus Res, 1996 Dec, 46(1-2), 149 - 56
Mutational analysis of bacteriophage phi CTX cos site; Xiong G et al.; The DNA of phi CTX contains the gene ctx, located closely downstream of cos . This encodes for the pore-forming cytotoxin protein, CTX . phi CTX converts some Pseudomonas aeruginosa strains into CTX producers . After different periods of phi CTX infection, two distinct forms of phage DNA were isolated: circular DNA from bacterial cytosol and later linear DNA from phi CTX particles . When circular phi CTX DNA was transfected into the P . aeruginosa strains CF5 and E40, phi CTX was amplified and ctx expressed . phi CTX induced a protein fraction in CF5 cells that cleaved the 0.477 kb cos fragment at the cos site, indicating terminase activity . Deletion and point mutation variants of the cos DNA were prepared . Protein binding to DNA in vitro and competition experiments in vivo showed that portions of the cos site and its flanking sequences are differentially critical to the binding of phi CTX-induced proteins.

J Antimicrob Chemother, 1996 Dec, 38(6), 1031 - 40
Resistance to bismuth among gram-negative bacteria is dependent upon iron and its uptake; Domenico P et al.; Bismuth antimicrobial action is poorly understood . Many trivalent metals possess antibacterial activity, especially under low iron conditions . Protection of bacteria from the deleterious effects of bismuth and other trivalent metals was demonstrated in iron-fortified media . Near-equimolar quantities of Fe3+ neutralized the growth-inhibitory effects of 250 microM Bi3+ . Resistance to bismuth action also depended on the production of virulence-related siderophores . Escherichia coli, Aeromonas hydrophila or Pseudomonas aeruginosa producing aerobactin, amonabactin or pyoverdin respectively, were most resistant to Bi3+ . Enterochelin or pyochelin producers were less resistant to Bi3+, but more resistant than strains lacking siderophores . Purified pyoverdin restored Bi3+ resistance in a mutant lacking this siderophore, but not in one lacking the pyoverdin receptor . Bismuth-treated bacteria exhibited unique outer membrane proteins, similar in size to iron-repressible proteins . Thus, resistance to the inhibitory action of Bi3+ among Gram-negative bacteria is inversely related to iron concentration and strongly dependent on iron transport mechanisms . The data suggest that bismuth action is largely a nonspecific, competitive interference with iron-transport, related primarily to atomic valence Furthermore, resistance to Bi3+ among bacteria is predictive of virulence.

Microbiol Res, 1996 Dec, 151(4), 359 - 70
Molecular cloning and homology modeling of protocatechuate 3,4-dioxygenase from Pseudomonas marginata; Petersen E et al.; The genes that encode the alpha and beta subunits of protocatechuate 3,4-dioxygenase (3,4-PCD {EC 1.13.11.3}) were cloned from a Pseudomonas marginata genomic library . These genes pcaG and pcaH, were found when screening the library for hydrolase genes . The two open reading frames of the PCD genes could be identified adjacent to an esterase gene by sequence homology . A 1.7-kb KpnI/ApaI fragment, carrying pcaG and pcaH, was subcloned and the genes were functionally expressed in Escherichia coli . The deduced amino acid sequence shows high homology to previously determined amino acid sequences of bacterial protocatechuate 3,4-dioxygenases . A homology model based on the available crystal structure of the protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa shows high similarity with the binding and catalytic sites.

Arch Dis Child, 1996 Dec, 75(6), 498 - 501
Relationship between disease severity and inflammatory markers in cystic fibrosis; Koller DY et al.; To evaluate the clinical use of measuring neutrophil, lymphocyte, and eosinophil activities, serum myeloperoxidase (MPO), soluble interleukin-2 receptors (sIL-2R), and eosinophil cationic protein (ECP) were measured in 98 patients with cystic fibrosis and in 85 healthy children . Serum concentrations of MPO, sIL-2R, and ECP were increased in patients with cystic fibrosis (median 807 micrograms/l, 4452 pg/ml, 48.8 micrograms/l, respectively) compared with the controls (median 319 micrograms/l, 2743 pg/ml, 9.4 micrograms/l) . ECP concentrations, but not serum MPO or sIL-2R, were significantly related to disease severity assessed by the Shwachman-Kulczycki score and by pulmonary function (forced expiratory volume in one second % predicted) . Neither ECP nor sIL-2R was influenced by Pseudomonas aeruginosa infection, acute pulmonary exacerbation, or atopy . Serum MPO, however, was strongly correlated with acute pulmonary exacerbation . In the light of these findings the measurement of serum ECP might thus be used for clinical monitoring and for assessing disease severity in cystic fibrosis . The measurement of serum MPO and sIL-2R did not correlate with the disease severity.

Kansenshogaku Zasshi, 1996 Dec, 70(12), 1271 - 8
{Pillin structural gene and bacterial adhesion to cultured cell of Pseudomonas aeruginosa isolated from clinical materials}; Nakazawa Y; Thirty-three stocks of Pseudomonas aeruginosa which is an important etiologic agent of opportunistic infections were clinically isolated . The pillin structural gene pilA of the stocks were amplified by polymerase chain reaction (PCR) and classified into 3 groups; 2000 bp (16 stocks; 48.5%), 1300 bp (11 stocks; 33.3%), 550 bp (6 stocks; 18.2%) . The adhesiveness of the stocks to cultured human lung cancer origin calu-1 was also determined, their adhesion rate per cell were 39.2%, 24.8%, 22.1% in average respectively . Thus clinically most common 2000 pb group is remarkably easier to adhere to calu-1 . Serotypes of the strains were examined to reveal the difference of the distribution that F . G . I types were dominant in 2000 pb group, but E types were major in 1300 bp and 550 bp groups . These data suggest that the gene arrangement of pilA influences adhesiveness to cultured cell and antigenicity of bacteria.

Appl Microbiol Biotechnol, 1996 Dec, 46(5-6), 638 - 46
Adding sodium dodecyl sulfate and Pseudomonas aeruginosa UG2 biosurfactants inhibits polycyclic aromatic hydrocarbon biodegradation in a weathered creosote-contaminated soil; Deschenes L et al.; The effect of two anionic surfactants was assessed during biodegradation of 13 of the 16 USEPA priority polycyclic aromatic hydrocarbons (PAH) in a wood-preserving soil contaminated with creosote and pentacholorophenol for a period of at least 20 years . Sodium dodecyl sulfate (SDS) and biosurfactants from Pseudomonas aeruginosa UG2 were utilized at concentrations of 10, 100 and 500 micrograms/g soil . Because both surfactants are readily biodegradable, the microcosms received a fresh spike of surfactant every 2 weeks . Biodegradation of aged PAH residues was monitored by GC/MS for a period of 45 weeks . Results indicated that the biodegradation of the three-ring PAH was rapid and almost complete but was slowed by the addition of 100 micrograms/g and 500 micrograms/g chemical surfactant . Similarly, at the same concentrations, the two surfactants significantly decreased the biodegradation rate of the four-ring PAH . In this case, the inhibition was more pronounced with SDS . High-molecular-mass PAH (more than four rings) were not biodegraded under the test conditions . It was suggested that the preferential utilization of surfactants by PAH degraders was responsible for the inhibition observed in the biodegradation of the hydrocarbons . The high biodegradability and the inhibitory effect of these two surfactants would have a significant impact on the development of both above-ground and in situ site reclamation processes.

Appl Microbiol Biotechnol, 1996 Dec, 46(5-6), 549 - 53
Evaluation of the biological containment system based on the Escherichia coli gef gene in Pseudomonas aeruginosa W51D; Soberon-Chavez G; The cell-killing gef gene was introduced, under the control of the lac promoter and as part of a transposon, into Pseudomonas aeruginosa W51D, a strain able to degrade branched-chain alkylbenzene sulfonates . Only 1% of the cells that inherited the transposon (Tngef) showed a conditional lethal phenotype, and this phenotype was lost at a high frequency without apparent loss of the tetracycline resistance encoded by the transposon . Southern blot analysis of two W51D::Tngef derivatives that expressed the cell-killing function showed multiple insertions of the transposon . These data suggest that Gef protein is able to kill P . aeruginosa W51D, but it seems that the level of resistance to Gef toxin in this stain is higher than that of previously reported bacteria, and that the expression of multiple copies of the gef gene is necessary to attain cell death . The higher level of resistance does not seem to be particular to strain W51D since two other P . aeruginosa strains analyzed (PAO2003 and ATCC 9027) also presented a small proportion of cells expressing the conditional lethal phenotype when they inherited the Tngef transposon.

Trends Microbiol, 1996 Dec, 4(12), 490 - 4
Pseudomonas aeruginosa lipopolysaccharides and pathogenesis; Goldberg JB et al.; Pseudomonas aeruginosa lipopolysaccharide (LPS) plays a key role in pathogenesis . In acute infections, a smooth LPS protects the organism from complement-mediated killing and, during chronic lung infections, an altered rough LPS helps the organism evade host defense mechanisms.

Acta Paediatr, 1996 Dec, 85(12), 1426 - 32
Essential fatty acid deficiency and predisposition to lung disease in cystic fibrosis; Lloyd-Still JD et al.; Essential fatty acid (EFA) deficiency is a predisposing factor for pulmonary infection with Staphylococcus aureus and Pseudomonas aeruginosa, the two major pathogenic microorganisms in cystic fibrosis (CF) . OBJECTIVE: The goal of this study was to investigate the essential fatty acid status of CF patients from infancy to 20 years old . MATERIALS AND METHODS: Plasma fatty acid profiles for phospholipid (PL) were determined for cord (n = 6), 4 months (n = 40), 16 months (n = 25), 3 y (n = 8), 5-10 y (n = 10), and 10-20 y (n = 10) aged CF patients and compared to their respective control; cord (n = 22), 1-36 months (n = 38) and adult (n = 100) . Significance was established by Student's t-test (p < 0.05) . RESULTS: The plasma PL fatty acid profile for all CF patients, except cord, revealed consistent deficiency in omega 3 and omega 6 EFAs . These deficiencies were most marked at infancy and more pronounced for patients with meconium ileus . CONCLUSIONS AND RELEVANCE: EFA deficiency may contribute to the predisposition of CF infants to develop respiratory disease and to the excess cytotoxic activity found in bronchoalveolar lavage fluid at 2 months of age in the majority of screened infants.

Indian J Med Res, 1996 Dec, 104, 342 - 8
A preliminary study of fingerprinting of Pseudomonas aeruginosa by whole cell protein analysis by SDS-PAGE; Khan FG et al.; Forty two strains of Pseudomonas aeruginosa isolated from bronchoalveolar lavage fluid from intubated patients admitted to the Intensive care unit in AIIMS between December 1993 to June 1994 were included in the study . After obtaining typical biochemical profile, antimicrobial susceptibility was performed against ceftazidime, amikacin, gentamicin, ampicillin, cefotaxime and ciprofloxacin . Pyocin typing of these 42 strains was performed by scrape and streak method using 22 indicator strains . Forty strains could be typed showing excellent discrimination but on repeated testing the group designation changed indicating that the system had low reproducibility . SDS-PAGE of whole cell protein profile indicated the presence of 45 protein bands of different molecular weights, individual isolates had 37 to 42 protein band ranging in molecular weight from 340 kDa to 14.3 kDa . On the basis of Dice index of similarity the strains could be grouped into 20 types . Since all strains could be typed, the system has adequate typability . Similar results were obtained on repeated testing indicating good reproducibility.

Chest, 1996 Dec, 110(6 Suppl), 256S - 260S
Opportunities for the use of aerosolized alpha 1-antitrypsin for the treatment of cystic fibrosis; Allen ED; Cystic fibrosis (CF), the most common lethal genetic disease affecting the white population, owes its morbidity and mortality primarily to the devastating effects of chronic inflammation and infection within the pulmonary airways . It has become increasingly recognized that the host's response to Pseudomonas species and Staphylococcus aureus infection plays a paramount role in CF lung destruction and eventual development of respiratory insufficiency . A massive pulmonary influx of neutrophils, and accompanying excessive levels of neutrophil elastase (NE), can be detected in the bronchoalveolar fluid of even very young children with CF . The excess of NE adversely affects the CF airways by enhancing mucus secretion, directly injuring airway tissues, exacerbating the inflammatory process by attracting more neutrophils, and derailing opsonization and elimination of bacterial pathogens, particularly Pseudomonas aeruginosa . Neutralization of excess NE by delivering supplemental alpha 1-antitrypsin to the airways via aerosolization represents an exciting new potential therapy for CF lung disease.

Int J Pept Protein Res, 1996 Dec, 48(6), 539 - 52
NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1 . Identification of a beta-turn; Campbell AP et al.; The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional 1H NMR techniques . Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data . Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides {McInnes, C., et al . (1993) Biochemistry 32, 13432-13440; Campbell, A.P., et al . (1995) Biochemistry 34, 16255-16268} reveals the common structural motif of a beta-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind . The importance of this conserved beta-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.

Clin Nephrol, 1996 Dec, 46(6), 394 - 401
Transfer of cytokine-inducing bacterial products across hemodialyzer membranes in the presence of plasma or whole blood; Pereira BJ et al.; Cytokine production by peripheral blood mononuclear cells (PBMC) is a sensitive indicator of cytokine-inducing substances which may cross from contaminated dialysate into the blood compartment . The objective of this study was to compare the transfer of cytokine-inducing substances from dialysate contaminated with a culture filtrate from Pseudomonas aeruginosa across dialyzers with low (hemophan) or intermediate ultrafiltration coefficients (modified cellulose triacetate, CTA), under conditions where either 10% plasma or whole blood was circulated in the blood compartment . Eight paired experiments of in vitro dialysis were carried out at 37 degrees C using a countercurrent recirculating loop dialysis circuit with either a new CTA or hemophan dialyzer . 10% plasma in standard tissue culture medium was circulated through the blood compartment and bicarbonate dialysate was circulated in the dialysate compartment . The dialysate was challenged sequentially by log-fold dilutions (10(2), 10(3) or 10(4)) of a Ps . aeruginosa culture filtrate . Samples were drawn from the blood compartment 5 and 15 minutes after each challenge and incubated with suspensions of PBMC in the absence or presence of polymyxin B, in order to block endotoxin . After 24 h at 37 degrees C, total interleukin-1 alpha (IL-1 alpha) was measured by RIA . Although the dialysate contained potent cytokine-inducing substances, there was no significant IL-1 alpha production by PBMC incubated with the plasma mixture from the blood compartment in the majority of experiments with both dialyzers and with each of the three dilutions of the bacterial challenge . Eight experiments were also performed with CTA dialzyers using heparinized whole blood in the blood compartment . Samples of whole blood and dialysate were drawn at baseline, after one hour of dialysis with uncontaminated dialysate and 15 minutes and three hours after dialysis with dialysate contaminated with Ps . aeruginosa filtrate . There was no significant IL-1 alpha production by PBMC isolated from the whole blood 1 h after dialysis with uncontaminated dialysate, and 15 min and 2 h after adding the Ps . aeruginosa filtrate to the dialysate side . In contrast, production of IL-1 alpha by PBMC from the same donors incubated with samples from the dialysate were 263 +/- 50, 1074 +/- 306, 2333 +/- 774 and 2602 +/- 702 pg/2.5 x 10(6) PBMC, respectively at the same four time points . These data suggest that although the Ps . aeruginosa culture filtrate present in the dialysate was a potent inducer of IL-1 alpha, neither dialyzer permitted transfer of cytokine inducing substances from the dialysate into the blood compartment.

J Chemother, 1996 Dec, 8(6), 420 - 4
Transferable resistance to specific antibiotics in nosocomial strains of Pseudomonas aeruginosa from two teaching hospitals; Blahova J et al.; We describe the transfer of resistance to kanamycin, carbenicillin and cephaloridine to a recipient strain of Escherichia coli K-12 No . 3110 from three strains of Pseudomonas aeruginosa out of 146 strains tested in 1995 . The P.aeruginosa No . 201, 203, 208 donor strains were isolated from patients in the University Clinics in Frankfurt, Germany . They were resistant to most beta-lactam antibiotics including cephalosporins of the 1st, 2nd and 3rd generation, imipenem, meropenem and aztreonam . They transferred kanamycin, carbenicillin and cephaloridine resistance determinants to recipient strain E.coli K-12 3110 . These determinants were accompanied in strain P . aeruginosa No . 203, with a transfer of ceftazidime resistance determinants and in P.aeruginosa No . 208 with transfer of cefotaxime, ceftazidime and aztreonam resistance determinants . Transfer of antibiotic resistance was also studied in 13 nosocomial strains of P.aeruginosa collected for their ceftazidime and/or imipenem resistance in a large Teaching Hospital in Ostrava, Czech Republic . Six of these strains transferred carbenicillin and/or cephaloridine resistance to the E.coli K-12 3110 recipient strain . Resistance to kanamycin and cefotaxime was also co-transferred with carbenicillin and cephaloridine determinants . Ceftazidime, imipenem or ofloxacin resistance was not transferred and is thus, most probably, of chromosomal origin.

J Chemother, 1996 Dec, 8(6), 411 - 5
In vitro efficacy of levofloxacin alone or in combination tested against multi-resistant Pseudomonas aeruginosa strains; Flynn CM et al.; Levofloxacin, the S(-) isomer of ofloxacin, demonstrates in vitro activity against Pseudomonas aeruginosa . To further characterize this activity, levofloxacin was tested against three populations of recent clinical isolates categorized by their resistance patterns to several other anti-pseudomonal agents . Results demonstrate the minimum inhibitory concentrations (MICs) for levofloxacin were generally two- to fourfold higher than for ciprofloxacin . Higher fluoroquinolone MICs were associated with MIC increases in other drugs . Levofloxacin demonstrated cross resistance against ciprofloxacin-resistant strains . Combinations of levofloxacin and several codrugs revealed that the majority of evaluable interactions demonstrated indifferent action . Levofloxacin exhibited enhanced activity (additive or degrees of synergy) principally with piperacillin, aztreonam, or ceftazidime . The synergy and additive rate (21 to 30%) compared favorably with the enhanced interactions observed with gentamicin combined with piperacillin or ceftazidime (27 to 30%) . Levofloxacin activity against P . aeruginosa was most comparable to that of ciprofloxacin, which was applicable against > 90% of strains found to be resistant to other classes of antimicrobial agents.

Mol Microbiol, 1996 Dec, 22(5), 1005 - 12
Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients; Wang J et al.; Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised individuals and those with cystic fibrosis (CF) . In CF patients, the secretion of abnormally high amounts of mucus into the airways contributes to their susceptibility to infection by P . aeruginosa . To identify virulence genes of P . aeruginosa that are important in infection of CF patients, an in vivo selection system (IVET) was used to identify promoters that are specifically inducible by respiratory mucus derived from CF patients . Three genetic loci that are highly inducible by the mucus were identified . One of them is a well-characterized virulence gene (fptA), encoding the receptor for pyochelin, which is a P . aeruginosa iron siderophore . Induction of the fptA gene by mucus is suppressed by the addition of exogenous iron, demonstrating that the mucus is an iron chelator and generates an iron-deficient environment in CF lungs . Therefore, as a part of the host-defence mechanism, the mucus could also be responsible for induction of iron-regulated virulence factors of bacterial pathogens . The second locus, np20, encodes a peptide that shares sequence homology to a number of transcriptional regulators . An identical locus was previously identified to be inducible in vivo during infection of mice and was shown to be important in bacterial virulence in a neutropenic-mouse infection model . The third locus, designated migA (mucus inducible gene), was sequenced and found to encode a 299-amino-acid peptide which is homologous to glycosyltransferases of other bacteria, and is involved in the biosynthesis of lipopolysaccharides or exopolysaccharides . Inducibilities of the np20 and migA genes are not affected by iron and the exact nature of the inducing signals in the mucus is not known . The possible implications of the migA inducibility by respiratory mucus is discussed in relation to the P . aeruginosa infection in CF.

J Bacteriol, 1996 Dec, 178(24), 7322 - 5
Anaerobic production of alginate by Pseudomonas aeruginosa: alginate restricts diffusion of oxygen; Hassett DJ; Pseudomonas aeruginosa produced alginate and elevated algD (encoding GDPmannose 6-dehydrogenase) transcription under strict anaerobic conditions, especially when using nitrate as a terminal electron acceptor . Purified alginate added to bacterial suspensions caused a decrease in growth, suggesting that alginate contributes to oxygen limitation for the organism and likely for patients afflicted with the inherited autosomal disease cystic fibrosis.

J Bacteriol, 1996 Dec, 178(24), 7120 - 8
Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis; Sundin GW et al.; We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism . Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P . aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate . In this study, we isolated four mutations in ndk (Ala-14-->Pro {Ala14Pro}, Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm . We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production . After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active . However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20 . Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study . All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis . Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis . The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions . These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as critical to this process.

Infect Immun, 1996 Dec, 64(12), 5417 - 20
Adhesion of Pseudomonas aeruginosa to respiratory mucins and expression of mucin-binding proteins are increased by limiting iron during growth; Scharfman A et al.; The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cystic fibrosis . Local factors in the respiratory tract, such as osmolarity or iron concentration, might influence this colonization . In the present work, we have observed that overall levels of adhesion of two nonmucoid, nonpiliated strains of P . aeruginosa, 1244-NP and PAK-NP, to human airway mucins were higher when these strains were grown in a minimal medium of low osmolarity (M9) than when they were grown in a rich medium of higher osmolarity (tryptic soy broth {TSB}) . However, increasing the NaCl concentration of M9 to increase the osmolarity did not modify the level of binding . In order to find out whether these differences were due to variations in nutrients, the influence of iron concentration was investigated: the levels of binding of the two strains increased after TSB was depleted of iron and decreased after iron was added to M9 . Since the outer membranes from the two strains have been shown to contain proteins reacting with human bronchial mucins, we compared the mucin-binding proteins expressed by the two strains grown in different media . When the nonpiliated strains 1244-NP and PAK-NP were grown in the different media, previously observed mucin-binding bands were detected in the 42- to 48-kDa range but additional mucin-binding bands in the 77- to 85-kDa range were detected when these strains were grown in M9 or iron-deprived TSB . These results demonstrate that the adhesion of P . aeruginosa and the expression of mucin-binding proteins in the outer membranes of nonpiliated P . aeruginosa are affected by the iron content of the medium in which the bacteria are grown and not by the osmolarity.

Infect Immun, 1996 Dec, 64(12), 4984 - 92
Lung phagocyte bactericidal function in strains of mice resistant and susceptible to Pseudomonas aeruginosa; Morissette C et al.; The host response to Pseudomonas aeruginosa lung infection varies among inbred mouse strains . Mice of the BALB/c strain are resistant to P . aeruginosa lung infection, whereas mice of the DBA/2 strain are susceptible . This phenotypic variation correlates with a difference in the magnitude of the inflammatory response induced early following infection . In order to determine whether the ability of lung phagocytic cells to kill P . aeruginosa plays a role in the host response to the infection, we measured the in vitro bactericidal activity of resident and inflammatory alveolar and interstitial macrophages, using a temperature-sensitive mutant of P . aeruginosa . Lung macrophages obtained from resistant and susceptible animals displayed similar bactericidal activities, suggesting that the ability of phagocytes to kill P . aeruginosa does not play a crucial role in the outcome of infection . The bactericidal activity of lung phagocytes was also assessed in vivo following endobronchial infection with the temperature-sensitive mutant of P . aeruginosa . Resistant mice showed a rapid influx of polymorphonuclear leukocytes (PMNs) to the bronchoalveolar space which was shortly followed by an efficient clearance of the bacteria . Susceptible mice had a delay in both the inflammatory response to P . aeruginosa and the initiation of bacterial clearance . Susceptible mice have been shown to have a defect in tumor necrosis factor alpha production when infected intratracheally with P . aeruginosa . Intratracheal instillation of tumor necrosis factor alpha to susceptible mice at the time of infection significantly improved the recruitment of PMNs to the site of infection without affecting the process of bacterial clearance . Overall, these results suggest that both recruitment of a high number of PMNs to the lungs and an efficient activation process of the phagocytes are crucial for the prompt clearance of P . aeruginosa.

Infect Immun, 1996 Dec, 64(12), 4922 - 7
Antiendotoxin activity of cationic peptide antimicrobial agents; Gough M et al.; The endotoxin from gram-negative bacteria consists of a molecule lipopolysaccharide (LPS) which can be shed by bacteria during antimicrobial therapy . A resulting syndrome, endotoxic shock, is a leading cause of death in the developed world . Thus, there is great interest in the development of antimicrobial agents which can reverse rather than promote sepsis, especially given the recent disappointing clinical performance of antiendotoxin therapies . We describe here two small cationic peptides, MBI-27 and MBI-28, which have both antiendotoxic and antibacterial activities in vitro and in vivo in animal models . We had previously demonstrated that these peptides bind to LPS with an affinity equivalent to that of polymyxin B . Consistent with this, the peptides blocked the ability of LPS and intact cells to induce the endotoxic shock mediator, tumor necrosis factor (TNF), upon incubation with the RAW 264.7 murine macrophage cell line . MBI-28 was equivalent to polymyxin B in its ability to block LPS induction of TNF by this cell line, even when added 60 min after the TNF stimulus . Furthermore, MBI-28 offered significant protection in a galactosamine-sensitized mouse model of lethal endotoxic shock . This protection correlated with the ability of MBI-28 to reduce LPS-induced circulating TNF by nearly 90% in this mouse model . Both MBI-27 and MBI-28 demonstrated antibacterial activity against gram-negative bacteria in vitro and in vivo against Pseudomonas aeruginosa infections in neutropenic mice.

J Immunol, 1996 Dec 1, 157(11), 5016 - 21
The roles of CD11/CD18 and ICAM-1 in acute Pseudomonas aeruginosa-induced pneumonia in mice; Qin L et al.; Neutrophil accumulation in response to Pseudomonas aeruginosa in the lungs is mediated through CD11/CD18 . This study determined the roles of CD11a, CD11b, and intercellular adhesion molecule (ICAM)-1 in P . aeruginosa-induced pneumonia and compared the function of ICAM-1 using Abs or ICAM-1 mutant mice . Anesthetized BALB/c mice pretreated with either Abs against CD11a, CD11b, ICAM-1, or rat IgG received intratracheal instillation of P . aeruginosa for 4 h . In other studies, ICAM-1 mutant and wild-type mice received either anti-ICAM-1 Ab or rat IgG followed by instillation of P . aeruginosa . The data show that Abs against CD11a, CD11b, and ICAM-1 in BALB/c mice inhibited neutrophil emigration by 79, 81, and 56%, respectively . ICAM-1 mutant mice showed no inhibition of neutrophil emigration compared with wild-type mice . Pretreatment with anti-ICAM-1 Ab inhibited neutrophil emigration in wild-type (129/SvxC57) mice by 67% but had no effect in ICAM-1 mutant mice, suggesting that the Ab was acting specifically through recognition of its Ag . We conclude that CD11a and CD11b are required for neutrophil emigration . The observed function of ICAM-1 varies depending on the method by which it is inhibited . Abs may overestimate function by altering other cellular functions or mutant mice may develop alternative pathways of emigration.

J Clin Microbiol, 1996 Dec, 34(12), 3226 - 9
Molecular epidemiology provides evidence of genotypic heterogeneity of multidrug-resistant Pseudomonas aeruginosa serotype O:12 outbreak isolates from a pediatric hospital; Bingen E et al.; Ribotyping randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis were used for the epidemiologic evaluation of eight Pseudomonas aeruginosa O:12 isolates obtained from eight children and two P . aeruginosa O:12 environmental isolates from a hematology ward . Randomly amplified polymorphic DNA analysis and pulsed-field gel electrophoresis were able to discriminate isolates that were indistinguishable by biochemical typing, O serotyping or ribotyping.

Curr Microbiol, 1996 Dec, 33(6), 347 - 51
Characterization and genetic mapping of phosphoglucoisomerase mutations in mannitol-negative mutants of Pseudomonas aeruginosa; Calligeros JE et al.; Phosphoglucoisomerase (pgi) mutations in a number of independently isolated mannitol-negative mutants of Pseudomonas aeruginosa PAO1 were mapped on the chromosome by plasmid FP5-mediated conjugation and by cotransduction with the generalized transducing phages G101 and F116L . Mutant allele pgi-9001 exhibited linkage to ilvB, C-9059 (46-85%), car-9003 (93-100%), and pur-9047 (70%), but not with met-9011, in FP5-mediated conjugational crosses . All known pgi mutations and several previously uncharacterized mannitol-negative mutations exhibited transductional linkage to two independent car mutations at frequencies ranging from 13% to 42% and 53% to 99%, in transductional crosses mediated by phages G101 and F116L respectively . These pgi and mannitol-negative mutations also were cotransducible at very low frequencies (<1%) with two independent ilv mutations . Cotransduction of the car and ilv loci could not be detected . These data suggest the location of pgi within the first minute of the P . aeruginosa chromosome closely linked to the car marker and probably between the ilv and car loci . All of the mannitol-negative mutations that exhibited linkage to the car and ilv loci were characterized as pgi mutations by enzyme assays . A phenotypically similar, mannitol-negative mutant was shown to contain a mutation in glucose-6-phosphate dehydrogenase (zwf-9012) that maps to a different region on the chromosome.

FEBS Lett, 1996 Nov 4, 396(2-3), 243 - 7
Specific photoaffinity labelling of a ferripyoverdin outer membrane receptor of Pseudomonas aeruginosa; Ocaktan A et al.; In order to identify and characterize the receptors involved in pyoverdin-mediated iron transport in Pseudomonas aeruginosa ATCC 15692, a photoactivatable siderophore has been synthesized . In the dark, this probe is stable and is able to promote iron transport at the same rate as the native pyoverdin . Under irradiation at 312 nm, the molecule is photodecomposed and a clear inhibition of the iron transport is observed . With the radioactive form of this photoactivatable probe, we were able to visualize on a SDS-PAGE gel a labelled protein of approximately 90 kDa molecular mass, which is very likely the FpvA receptor or a yet unknown pyoverdin receptor.

FEBS Lett, 1996 Nov 4, 396(2-3), 213 - 7
Genetic diversity of flagellins of Pseudomonas aeruginosa; Spangenberg C et al.; Physical genome analysis of the virulence-associated fliC locus in 20 Pseudomonas aeruginosa strains by mapping and sequencing revealed groups of heterologous a-type (1164 bp; 1185 bp) and highly conserved b-type (1467 bp) flagellin genes . Whereas only two synonymous nucleotide substitutions were detected in eight b-type fliC sequences, the 12 a-type sequences exhibited 57 nucleotide substitutions, of which 39 occurred within a variable central region . Although a-type and b-type flagellins differ by 35% in their primary structure, they share strong homology in their predicted features, implying that the polymorphic proteins fold into similar structures during polymerization of the flagella.

Enferm Infecc Microbiol Clin, 1996 Nov, 14(9), 519 - 23
{Pseudomonas spp . infections in patients with HIV infections}; Payeras A et al.; BACKGROUND: The aim of this study was to describe the characteristics of the infections by Pseudomonas spp . observed in patients with HIV infection in Spain . METHODS: A retrospective study was performed of the isolations of Pseudomonas spp . in microbiologic samples of patients with HIV infection in three hospitals from Mallorca, Spain, since 1986 . RESULTS: Twenty-nine patients with some positive culture for Pseudomonas were reviewed . In 20 cases the infection presented in advanced stages of the disease when the patient fulfilled AIDS criteria . The most frequent foci in both community acquired and nosocomial infection was respiratory (16 and 3 cases, respectively) . Fifty percent of the cases presented bacteremia . The classically described predisposing factors for infection by this germ were presented in 19 patients . Pseudomonas aeruginosa was the most frequently isolated type (22 cases) . Only 5 patients received the appropriate treatment on admission . Clinical cure was achieved in 23 patients, with recurrence being observed in 10 . Five patients died in relation to the infection . CONCLUSIONS: Infections by Pseudomonas spp . in Spain appear to have increased in frequency in patients with HIV infection in the last decade . These infections appear in advanced phases of the disease and mainly involve the lung, with high rates of bacteremia and a high number of recurrence . Empiric treatment of patients with advanced HIV infection with suspicion of bacterial infection should include antipseudomonic drugs.

J Biochem (Tokyo), 1996 Nov, 120(5), 915 - 21
Substitutions of Ser for Asn-163 and Pro for Leu-264 are important for stabilization of lipase from Pseudomonas aeruginosa; Shinkai A et al.; The lipase gene from Pseudomonas aeruginosa was randomly mutated by error-prone PCR to obtain thermostable mutants, followed by screening for thermostable mutant lipases . Out of about 2,600 transformants, four thermostable clones were obtained . Their nucleotide sequences showed that they had two or three amino acid substitutions . Analysis of the thermal stabilization of these mutant lipases indicated that Asn-163 to Ser and Leu-264 to Pro mutations were essential for the increased stability of the lipase . We expressed a mutant lipase (StLipA-5) having only the Asn-163 to Ser mutation and another (StLipA-6) having only the Leu-264 to Pro mutation in P . aeruginosa PAO1161, purified them, and then confirmed that the temperature which causes a 50% decrease in the activity of the non-treated enzyme on treatment for 30 min was increased by 1.5 and 3 degrees C, respectively, compared to the wild-type enzyme . However, the thermal stability of the mutant lipase (StLipA-7) having both mutations was increased only by 2.5 degrees C . These mutant lipases were stabilized through a decrease in activation entropy . Kinetic studies showed that the Kcat/K(m) values of StLipA-5, StLipA-6, and StLipA-7 were decreased by 14.4, 52.9, and 26.0%, respectively . Interestingly, the pH-stabilities of StLipA-6 and StLipA-7 were also increased, especially at alkaline pH . Based on these results, the tertiary structure and mechanism of stabilization of the lipase were discussed.

J Am Optom Assoc, 1996 Nov, 67(11), 676 - 80
Survival of contaminating bacteria in over-the-counter artificial tears; Harris MG et al.; BACKGROUND: The use of OTC artificial tears is becoming increasingly widespread . Unintentional contamination of artificial tear products potentiates a risk of developing bacterial keratitis or exacerbating an existing eye infection . Presently, no published study has investigated recovery from bacterial contamination of artificial tear products . METHODS: Four different over-the-counter (OTC) artificial tear products were contaminated with two types of bacteria . Staphylococcus aureus and pseudomonas aeruginosa were analyzed for the survivability of the microorganisms at various times following contamination . RESULTS: In this cross-sectional study, it was found that all the preserved brands showed a significant recovery rate from contamination compared to the preservative-free brand . After 9 hours, the preservative-free brand did not completely regain sterility after contamination with either bacteria . CONCLUSIONS: This study indicates that preserved artificial tears recover from bacterial contamination at a significantly faster rate than preservative-free products . It is likely that the recovery from bacterial contamination is not solely attributed to the preservative, but that other variables may contribute.

J Antimicrob Chemother, 1996 Nov, 38(5), 853 - 8
The effect of aminoglycoside-induced adaptive resistance on the antibacterial activity of other antibiotics against Pseudomonas aeruginosa in vitro; Barclay ML et al.; The effect of gentamicin-induced adaptive resistance on the antibacterial activity of six non-aminoglycoside antibiotics was studied . Adaptive resistance was induced in Pseudomonas aeruginosa in a dynamic in-vitro model of infection . The bactericidal effect of ceftazidime, imipenem, aztreonam, ciprofloxacin, and piperacillin was not altered in the presence of adaptive resistance but the effect of rifampicin was increased.

J Antimicrob Chemother, 1996 Nov, 38(5), 809 - 18
Aerosolization of imipenem/cilastatin prevents pseudomonas-induced acute lung injury; Hashimoto S et al.; Aerosolization of imipenem/cilastatin was compared with continuous intravenous infusions of the antibiotic for pharmacokinetic/pharmacodynamic analysis . The concentrations of imipenim/cilastatin in bronchoalveolar lavage fluids (BAL) obtained from rats exposed to the aerosolized antibiotic were significantly greater than the concentrations in BAL in the rats that had received intravenous infusions of imipenem/cilastatin . The two methods of antibiotic delivery were compared for their effects on bacterial-induced lung injury in rats that had Pseudomonas aeruginosa instilled into their airspaces . The aerosolization of antibiotic was associated with significantly decreased bacterial-induced lung injury . The high concentrations of antibiotic in the airspaces secondary to aerosolization appears to kill bacteria more quickly and preserve lung epithelial and endothelial integrity better than systemic delivery of the same antibiotic.

Chemotherapy, 1996 Nov-Dec, 42(6), 426 - 31
Activities of piperacillin, ceftazidime, cefepime and cefpirome against Pseudomonas aeruginosa strains with intrinsic ticarcillin resistance; Bert F et al.; The in vitro activities of cefepime and cefpirome against 44 intrinsically ticarcillin-resistant strains of Pseudomonas aeruginosa were compared to their activities against 20 ticarcillin-susceptible strains by MIC determination and the disk test . Time-killing curves were constructed for piperacillin, ceftazidime, cefepime and cefpirome against two susceptible and two resistant strains . The activities of cefepime and cefpirome against the resistant strains were impaired, and most of the strains were of intermediate sensitivity to these agents . The time-killing curves of the four beta-lactams were similar, with a modest decline in viable cell counts over the first 6 h followed by regrowth . There was no difference between the susceptible and resistant strains.

Eur Respir J, 1996 Nov, 9(11), 2207 - 14
Genotype-phenotype relationships in a cohort of adult cystic fibrosis patients; Hubert D et al.; In cystic fibrosis (CF), relationships between genotype and phenotype have been shown for pancreatic status but not for pulmonary disease . One hundred and ten adult CF patients were classified according to the expected effect of their mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein: Group 1 (n=48) included deltaF508 homozygotes; Group 2 (n=26), patients with two "severe" mutations and no expected CFTR production; Group 3 (n=17), patients with expected partly functional CFTR corresponding to at least one "mild" mutation; Group 4 (n=19), patients with no mutation identified or only one identified "severe" mutation . As compared to Groups 1 and 2: patients from Groups 3 and 4 had higher arterial oxygen tension (Pa,O2) (9.5+/-1.9 and 9.9+/-1.5 vs 8.8+/-1.5 and 8.3+/-1.7 kPa, respectively p<0.02); and a slower decline in their pulmonary function, estimated by the mean annual loss in forced vital capacity (FVC) (1.2+/-1.0 and 1.5+/-1.1 vs 2.0+/-0.9 and 2.2+/-1.0%, respectively; p<0.01) and in forced expiratory volume in one second (FEV1) (1.7+/-1.1 and 1.9+/-1.3 vs 2.6+/-1.0 and 2.8+/-1.0%, respectively; p<0.005) . They had fewer episodes of colonization of the airways by Pseudomonas aeruginosa, and colonization occurred at a more advanced age (median age 25 and 19 vs 15 and 17 yrs, respectively; p<0.01) and required fewer intravenous antibiotic courses (p<0.01) . Pancreatic insufficiency was less frequent in Groups 3 (23%) and 4 (63%) than in Groups 1 (100%) and 2 (96%) . This study suggests that the phenotype of adult cystic fibrosis patients, including the severity of the lung disease, is related to the severity of the cystic fibrosis transmembrane conductance regulator mutations.

Am J Physiol, 1996 Nov, 271(5 Pt 1), L838 - 43
Dimethyl sulfoxide decreases interleukin-8-mediated neutrophil recruitment in the airways; Massion PP et al.; In this study, we investigated the role of dimethyl sulfoxide (DMSO) in inhibiting interleukin-8 (IL-8)-mediated neutrophil recruitment induced by Pseudomonas aeruginosa (PA) bacterial supernatant . First, we tested whether DMSO could inhibit IL-8 production induced by PA in human bronchial epithelial (16-HBE) cells in vitro . In these cells, exposure to PA or H2O2 induced IL-8 production dose dependently, an effect that was inhibited by 1% DMSO at both the protein and RNA level . Second, we tested whether DMSO could block the recruitment of neutrophils induced by PA in a bypassed segment of dog trachea in vivo . PA supernatant was placed in the tracheal segment for 6 h in four dogs, and neutrophil recruitment and IL-8 concentrations were measured in the superfusate . DMSO prevented the recruitment of neutrophils and IL-8 production induced by PA time dependently . The results suggest that DMSO may play an anti-inflammatory role in the airway by inhibiting IL-8 production in epithelial cells.

Mol Microbiol, 1996 Nov, 22(3), 481 - 95
Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster; Burrows LL et al.; Pseudomonas aeruginosa co-expresses A-band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B-band LPS, a heteropolymer with a repeating unit of 2-5 sugars which is the serotype-specific antigen . The gene clusters for A- and B-band biosynthesis in P . aeruginosa O5 (strain PAO1) have been cloned previously . Here we report the DNA sequence and molecular analysis of the B-band O-antigen biosynthetic cluster . Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz (rol), wzy (rfc), and wbpA-wbpN . A further 3 ORFs not thought to be involved with LPS synthesis were identified (hisH, hisF, and uvrB) . Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O-antigen serogroup . In contrast, wbpM and wbpN are common to all 20 serotypes of P . aeruginosa . Although wbpM is not serogroup-specific, knockout mutations confirmed it is necessary for O5 O-antigen biosynthesis . A novel insertion sequences, IS 1209, is present at the junction between the serogroup-specific and non-specific regions . We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P . aeruginosa O5 O-antigen biosynthesis.

Protein Sci, 1996 Nov, 5(11), 2248 - 54
Probing the structure and mobility of Pseudomonas aeruginosa azurin by circular dichroism and dynamic fluorescence anisotropy; Mei G et al.; The UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied . Two classes of mutants were examined . In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants 17S and F110S) . In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F, H35L, and H35Q . All these proteins showed two fluorescence lifetimes in the copper-containing form, but only one in the copper-free form . The lifetime of the latter derivatives was different from either those of the metal-bound samples, definitely ruling out the presence of apo-like species in the holo protein . Copper-free 17S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime . Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes . Time-resolved anisotropy of these two mutants as well as of wild-type (wt) protein showed two components (rotational times for wt < or = 200 ps and 7 ns, respectively) . These components were not affected significantly by copper removal in the case of wt protein . Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins . These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270-295 nm related to a relaxation of the structural constraint around the tryptophan . Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of 17S and F110S mutants . Furthermore, although the metal site geometry is rigidly kept in wt apo-azurin, it regains the native form only in the presence of the metal in the "core" mutants . This finding is important for the theory of entatic states in metalloproteins (Williams RJP, 1995, Eur J Biochem 234:363-381).

Clin Infect Dis, 1996 Nov, 23(5), 1109 - 16
Serum antibody concentrations of cytotoxin, exotoxin, A, lipopolysaccharide, protease, and elastase and survival of patients with Pseudomonas aeruginosa bacteremia; Baltch AL et al.; The relationship between survival and serum concentrations of antibody to cytotoxin, exotoxin A, lipopolysaccharide (LPS; IgM and IgG), protease, and elastase (determined by enzyme-linked immunosorbent assay) was studied in a group of 41 patients with Pseudomonas aeruginosa bacteremia . The lowest mean concentrations of antibody to cytotoxin (P < .05) and of IgG to LPS (P < .01) were noted in the patients who died within 48 hours of onset . The mean level of antibody to cytotoxin in patients who died 9-45 days or 5-22 months following onset was similar to that in controls, while in patients who survived > or = 24 months the level was higher than in controls (P < .01) . The mean level of IgG to LPS was highest in patients who survived > or = 24 months . The mean concentrations of antibody to the other antigens (except IgM to LPS) were higher in patients than in controls (P < .01) . No significant change occurred in any mean antibody concentrations over time . Administration of antipseudomonal antibodies, especially to cytotoxin and LPS, should be considered in the early stages of therapy for patients with P . aeruginosa bacteremia.

Clin Infect Dis, 1996 Nov, 23(5), 973 - 8
Evaluation of outcome for intubated patients with pneumonia due to Pseudomonas aeruginosa; Rello J et al.; Thirty consecutively intubated patients with pneumonia due to Pseudomonas aeruginosa (cases) were prospectively observed to establish the attributable mortality rate and the prognostic value of APACHE (Acute Physiological and Chronic Health Evaluation) II scores . Four cases did not receive accurate empirical therapy and were excluded from the study . APACHE II scores were calculated within 24 hours of admission (T0), at the time of the diagnosis of pneumonia (T1), and after 72 hours of therapy (T2) . The outcomes for these cases (n = 26) were compared with those for matched controls (n = 52) without pneumonia . Six cases died of causes directly related to pneumonia (group D) . Two cases whose conditions clinically improved died of cardiac complications, and 18 cases had clinical resolution (group R); however, only 15 of these cases were alive at discharge . The mean APACHE II score at admission was similar (P > .20) for group R, group D, and controls . In contrast, the mean score at T1 (15.40 +/- 6.07 vs . 20.83 +/- 4.66; P < .05) and the mean score at T2 (10.40 +/- 3.57 vs . 25.50 +/- 3.93; P < .01) differed significantly for groups R and D, respectively . The overall observed and predicted mortality rates among cases and controls were 42.3% and 28.1% and 28.8% and 28.7%, respectively, while the attributable mortality rate among cases was estimated to be 13.5% (95% confidence interval, 1.95%-25.04%) . We conclude that the attributable mortality rate among intubated patients with pneumonia due to P . aeruginosa is high . The APACHE II score at admission is not useful as a prognostic factor, while progression of organ dysfunction after the onset of pneumonia is an ominous sign.

EMBO J, 1996 Nov 1, 15(21), 5907 - 16
Transcription antitermination regulation of the Pseudomonas aeruginosa amidase operon; Wilson SA et al.; In vivo titration experiments have demonstrated a direct interaction between the Pseudomonas aeruginosa transcription antiterminator, AmiR, and the mRNA leader sequence of the amidase operon . A region of 39 nucleotides has been identified which is sufficient to partially titrate out the AmiR available for antitermination . Site-directed mutagenesis has shown that the leader open reading frame has no role in the antitermination reaction, and has identified two critical elements at the 5' and 3' ends of the proposed AmiR binding site which are independently essential for antitermination . A T7 promoter/RNA polymerase-driven system shows AmiR-mediated antitermination, demonstrating a lack of promoter/polymerase specificity . Using the operon negative regulator, AmiC, immobilized on a solid support and gel filtration chromatography, an AmiC-AmiR complex has been identified and isolated . Complex stability and molecular weight assayed by gel filtration alter depending on the type of amide bound to AmiC . AmiC-AmiR-anti-inducer is a stable dimer-dimer complex and the addition of the inducer, acetamide, causes a conformational change which alters the complex stability and either this new configuration or dissociated AmiR interacts with the leader mRNA to cause antitermination.

Clin Diagn Lab Immunol, 1996 Nov, 3(6), 727 - 32
Characterization of monoclonal antibody B7, which neutralizes the cytotoxicity of Pseudomonas aeruginosa exotoxin A; Shang HF et al.; A nontoxic Pseudomonas aeruginosa exotoxin A (PE), which has the carboxyl-terminal 38 amino acid residues of native PE deleted, was used as an antigen to immunize BALB/c mice, which were then challenged with native PE in order to raise monoclonal antibodies (MAbs) that can neutralize PE cytotoxicity . A murine MAb against PE, designated MAb B7, was established . MAb B7 was characterized in terms of its ability to neutralize PE cytotoxicity, epitope mapping, inhibition of PE receptor binding, and influence on cellular processing of PE and ADP-ribosylation activities . We found that MAb B7 could neutralize PE cytotoxicity in cell culture and in BALB/c mice . The epitope recognized by MAb B7 was mapped to the carboxyl-terminal amino acid residues 575 to 595 of PE . Consistent with the results of epitope mapping, MAb B7 did not block PE receptor-binding activity or the cellular processing of PE but strongly inhibited the ADP-ribosylating activity of PE . In addition, MAb B7 retained strong binding to PE even at pH 4.0, indicating that the complex of MAb B7 and PE is stable in the phagolysosome . On the basis of these observations, the neutralization of PE cytotoxicity by MAb B7 could be due to its binding to the carboxyl terminus of PE . As a result, MAb B7 may interfere with the interaction of the carboxyl-end amino acid residues REDLK of PE with cellular factors . However, we could not rule out the possibility that MAb B7 directly blocks the ADP-ribosylation activity of PE in the cytosol.

Antimicrob Agents Chemother, 1996 Nov, 40(11), 2488 - 93
A TEM-derived extended-spectrum beta-lactamase in Pseudomonas aeruginosa; Mugnier P et al.; A clinical strain of Pseudomonas aeruginosa, PAe1100, was found to be resistant to all antipseudomonal beta-lactam antibiotics and to aminoglycosides, including gentamicin, amikacin, and isepamicin . PAe1100 produced two beta-lactamases, TEM-2 (pI 5.6) and a novel, TEM-derived extended-spectrum beta-lactamase called TEM-42 (pI 5.8), susceptible to inhibition by clavulanate, sulbactam, and tazobactam . Both enzymes, as well as the aminoglycoside resistance which resulted from AAC(3)-IIa and AAC(6')-I production, were encoded by an 18-kb nonconjugative plasmid, pLRM1, that could be transferred to Escherichia coli by transformation . The gene coding for TEM-42 had four mutations that led to as many amino acid substitutions with respect to TEM-2: Val for Ala at position 42 (Ala42), Ser for Gly238, Lys for Glu240, and Met for Thr265 (Ambler numbering) . The double mutation Ser for Gly238 and Lys for Glu240, which has so far only been described in SHV-type but not TEM-type enzymes, conferred concomitant high-level resistance to cefotaxime and ceftazidime . The novel, TEM-derived extended-spectrum beta-lactamase appears to be the first of its class to be described in P . aeruginosa.

Eur J Pediatr, 1996 Nov, 155(11), 948 - 53
Tolerance, pharmacokinetics and efficacy of once daily amikacin for treatment of Pseudomonas aeruginosa pulmonary exacerbations in cystic fibrosis patients; Vic P et al.; Twenty cystic fibrosis patients aged 1.8-22 years (mean +/- SD: 9.6 +/- 4.8 years) with Pseudomonas aeruginosa pulmonary exacerbations were treated with amikacin (AM) (35 mg/kg/day in one daily 30 min infusion) associated with either ceftazidime (200 mg/kg/day in 3 i.v . injections) (n = 19) or imipenem (n = 1) at the same dose . Glomerular and tubular functions (creatinine clearance, 24-h proteinuria, beta 2 microglobulinuria, lysozymuria) and audiometry remained within normal ranges from day 0 to day 14 . A peak concentration of AM of 83 +/- 19 mg/l and a trough concentration of 0.8 +/- 0.5 mg/l were observed in blood while AM levels in sputum were above the minimal inhibitory concentration 50 from 30 min to 16 h . No serum accumulation of AM was observed during the treatment . From day 0 to day 14, the following changes were observed: weight/height ratio: 96%-100% (P < 0.001); daily energy intake: 111%-128% of RDA (P < 0.001); prealbumin: 195-290 mg/l (P < 0.001); forced vital capacity (FVC): 66%-81% (P < 0.01); forced expiratory volume in 1 s: 60%-75% (P < 0.01); forced expiratory flow between 25% and 75% of FVC: 42%-56% (P < 0.01); nocturnal SaO2 also improved significantly; cardiac rate decreased from 89 +/- 18/min to 76 +/- 16/min (P < 0.001); respiratory rate decreased from 31 +/- 15/min to 26 +/- 10/min (P < 0.05); inflammatory parameters (white blood cells, polymorphonuclear cells, erythrocyte sedimentation rate) also improved . CONCLUSION: Once daily amikacin administration associated with ceftazidime is well tolerated for the treatment of Pseudomonas aeruginosa pulmonary exacerbations in cystic fibrosis patients . Serum peak levels and diffusion in sputum are higher than with a conventional schedule.

Arch Otolaryngol Head Neck Surg, 1996 Nov, 122(11), 1209 - 13
Clinical characteristics and genotype analysis of patients with cystic fibrosis and nasal polyposis requiring surgery; Kingdom TT et al.; OBJECTIVE: To analyze the clinical characteristics and genotypes of patients with cystic fibrosis (CF) and nasal polyposis who require surgery . DESIGN: Cross-sectional analysis of a large patient database . SETTING: Data obtained from the National CF Patient Registry of the Cystic Fibrosis Foundation, Bethesda, Md . PATIENTS: Clinical and genotype data on 20198 patients with CF who were registered in 1992 and 1993 were analyzed . The study group (n = 815) consisted of patients with CF who had undergone surgical procedures for the treatment of nasal polyposis . The comparison group (n = 19383) comprised the remainder of the patients in the database . RESULTS: Statistical analysis revealed that patients with CF and nasal polyposis who required surgery had better pulmonary function (higher percent-predicted forced expiratory volume in 1 second and forced vital capacity), better nutritional status, a higher rate of Pseudomonas aeruginosa colonization, more office visits, more hospitalizations, and a higher rate of acute exacerbations per year (P < .001 for each) than did the comparison group . Among the patients who had mutation analysis performed, patients with nasal polyposis who required surgery were significantly associated with 2 specific genotypes: the delta-F508/delta-F508 (57.5% vs 49.9%, P = .01) and the delta-F508/G551D (12% vs 8%, P = .05) genotypes . CONCLUSIONS: Patients with CF and nasal polyposis who require surgery may constitute a clinical subgroup within the spectrum of the disease . These patients appear to have slightly better pulmonary function and nutritional status; yet, they seem to have a higher degree of health care utilization . The higher rate of P aeruginosa respiratory infection in this patient group suggests an association with the presence of nasal polyposis . Genotype analysis showed a higher prevalence of the delta-F508/delta-F508 and the delta-F508/G551D genotypes in this patient group.

Infect Immun, 1996 Nov, 64(11), 4842 - 5
Bactericidal activity of a synthetic peptide (CG 117-136) of human lysosomal cathepsin G is dependent on arginine content; Shafer WM et al.; The importance of individual amino acids in mediating the broad-spectrum bactericidal action of a 20-mer amphipathic, cationic peptide (CG 117-136) of human lysosomal cathepsin G was determined by using a single amino acid replacement strategy . This strategy revealed an important role for arginine because loss of any of the four arginine residues in CG 117-136 due to substitution with alanine, citrulline, or lysine residues resulted in a reduction of its bactericidal activity against both Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 33593 . However, the replacement of a single alanine residue in CG 117-136 with arginine, but not glutamic acid, enhanced the activity of CG 117-136 against both P . aeruginosa and S . aureus . The importance of certain bulky, nonpolar amino acids for the bactericidal activity of CG 117-136 was also evident, since their substitutions by alanine diminished bactericidal activity . Accordingly, contributions of hydrophobic amino acids and structural considerations of the guanidinium side chain of arginine are major determinants in the broad-spectrum antimicrobial action of CG 117-136.

Curr Microbiol, 1996 Nov, 33(5), 312 - 6
Methods for increasing survivability during storage of exponentially growing bacteria; Berger M et al.; A protocol has been developed for storing Gram (-) bacterial cells at 0 degrees C, which allows greater than 90% of stored cells to retain colony-forming ability for up to 60 days . The protocol, which yields essentially identical results when used with Escherichia coli or Pseudomonas aeruginosa, does not enhance survivability of Bacillus cereus . The greatest and longest survival is enjoyed when exponentially growing cells in minimal-glucose medium are deprived of carbon for about 9 h, supplemented with 750 microg/ml chloramphenicol, and immediately placed at 0 degrees C . By decreasing the period of carbon starvation from 9 to 5 h, or increasing the period of carbon starvation from 9 to 12 h, both the ultimate survival rate and kinetics of loss of culturability are affected . Survival enhancement induced by chloramphenicol is not similarly induced by kanamycin.

Gene, 1996 Oct 24, 177(1-2), 47 - 53
Genetic and sequence analysis of the cos region of the temperate Pseudomonas aeruginosa bacteriophage, D3; Sharp R et al.; The location and structure of the cos ends of bacteriophage D3, which infects Pseudomonas aeruginosa strain PAO, has been determined using a combination of deletion analysis, transposon mutagenesis, and sequencing directly off the phage DNA . Phage D3 was found to have 9-bp 3' cos ends, making it the first phage of a Gram-negative organism known to have 3' cos ends . A 700-bp region flanking the cos site was necessary for efficient transduction of D3 cosmid derivatives . This region was found to contain incomplete inverted repeat sequences flanking the cos site, along with adenine-rich repeats homologous to coliphage gama Ter binding sites . Possible IHF binding sites were also present.

Gene, 1996 Oct 17, 176(1-2), 55 - 9
Characterisation of the pvdE gene which is required for pyoverdine synthesis in Pseudomonas aeruginosa; McMorran BJ et al.; Pseudomonas aeruginosa (Pa) strain PAO synthesises a siderophore, pyoverdine (Pvd), when grown under conditions of iron starvation . Pvd consists of a dihydroxyquinoline group attached to an 8-amino-acid-residue peptide . DNA spanning at least 78 kb of the chromosome is required for Pvd synthesis, but to date only three genes involved in this process have been characterised . We report the characterisation of a fourth Pa gene, pvdE, which we show to be required for Pvd synthesis . The deduced amino acid sequence of PvdE indicates that the protein is a member of the ATP-binding-cassette (ABC) family of membrane transporter proteins, and this is the first example of the involvement of an ABC-type protein in siderophore synthesis.

Arch Intern Med, 1996 Oct 14, 156(18), 2121 - 6
Epidemiology and outcome of Pseudomonas aeruginosa bacteremia, with special emphasis on the influence of antibiotic treatment . Analysis of 189 episodes; Vidal F et al.; OBJECTIVE: To evaluate the trend in incidence of Pseudomonas aeruginosa bacteremia, underlying conditions of patients, mortality rate, and factors associated with poor outcome . PATIENTS AND METHODS: Medical charts of 189 consecutive episodes of P aeruginosa bacteremia, detected between January 1, 1991, and December 31, 1994, were prospectively evaluated . Associated risk factors, treatment, and outcome were recorded . RESULTS: Pseudomonas aeruginosa bacteremia represented 5.7% of the total number of bacteremias, 6.9% of nosocomial bacteremias, and 23.6% of nosocomial gram-negative bacteremias . There were 1.5 episodes per 1000 discharges . These numbers were slightly lower than those recorded at our hospital 10 years earlier . Human immunodeficiency virus infection was the most frequent underlying disease (28/189 {15%}) . Overall mortality was 18% (34/189) . The presence of fatal underlying disease (P < .001), surgery (P = .001), pneumonia (P = .02), and severe sepsis (P < .001) were associated with poor prognosis, the mortality of the patients with these variables being 28%, 28%, 47%, and 62%, respectively . The presence of inappropriate definitive antimicrobial treatment became an independent factor predictive of death (P = .04) only when the subset of patients with intravenous catheter-associated bacteremia was excluded from the analysis . The survival rate was no greater in patients who received 2 or more antibiotics active in vitro against P aeruginosa than in those who received only 1 . Neutropenia was not associated with increased mortality . The use of colony-stimulating factors did not affect the outcome of the neutropenic patients . CONCLUSIONS: The rate of P aeruginosa bacteremia is falling slightly at our hospital . The emergence of the human immunodeficiency virus epidemic has had a considerable impact on both epidemiology and mortality . The presence of severe underlying disease, surgery, pneumonia, and, especially, severe sepsis are associated with a poor outcome . With the exclusion of patients with intravenous catheter-associated P aeruginosa bacteremia, the administration of an appropriate antimicrobial therapy is essential to a good outcome . Treatment with 1 active antibiotic seems to be sufficient.

Gene, 1996 Oct 10, 175(1-2), 143 - 50
Functional expression of heterologous type 4 fimbriae in Pseudomonas aeruginosa; Watson AA et al.; Type 4 fimbriae are surface organelles produced by a wide range of bacterial pathogens . In Pseudomonas aeruginosa they are associated with a form of surface translocation known as twitching motility and have also been implicated as the receptor for a number of fimbrial-specific bacteriophages . The infrastructural machinery required for type 4 fimbrial biogenesis appears to be conserved as heterologous subunits from other species can be expressed in P . aeruginosa . All of these studies have, until now, been performed in non-functional Pseudomonas host strains which lack twitching motility . We have constructed isogenic mutants of two commonly studied wild-type P . aeruginosa strains, PAK and PAO1, by replacing the entire pilA gene which encodes the fimbrial subunit . Fimbrial expression and twitching motility were restored by complementation in trans with either the homologous or heterologous subunits from these strains, as well as that from another type 4 fimbriate species, Dichelobacter nodosus . The expression of different subunits allowed us to investigate the precise role that the individual subunit proteins contribute to bacteriophage infection by several fimbrial-specific bacteriophages . Sensitivity to bacteriophages B3cts and D3112cts was restored by the expression of any fimbrial subunit in both PAO1 and PAK cells, indicating that infection by these bacteriophages is fimbrial dependent but not fimbrial specific . In contrast, while sensitivity to the PAK-specific bacteriophage PO4 was restored by the expression of any fimbrial subunit in PAK cells, this did not occur in PAO1 cells except when expressing the PAK subunit . In all cases, the presence of fimbriae was absolutely required to allow a productive bacteriophage infection to occur.

Antimicrob Agents Chemother, 1996 Oct, 40(10), 2288 - 90
Multidrug efflux in intrinsic resistance to trimethoprim and sulfamethoxazole in Pseudomonas aeruginosa; Kohler T et al.; Pseudomonas aeruginosa possesses at least two multiple drug efflux systems which are defined by the outer membrane proteins OprM and OprJ . We have found that mutants overexpressing OprM were two- and eightfold more resistant than their wild-type parent to sulfamethoxazole (SMX) and trimethoprim (TMP), respectively . For OprJ-overproducing strains, MICs of TMP increased fourfold but those of SMX were unchanged . Strains overexpressing OprM, but not those overexpressing OprJ, became hypersusceptible to TMP and SMX when oprM was inactivated . The wild-type antibiotic profile could be restored in an oprM mutant by transcomplementation with the cloned oprM gene . These results demonstrate that the mexABoprM multidrug efflux system is mainly responsible for the intrinsic resistance of P . aeruginosa to TMP and SMX.

Rev Clin Esp, 1996 Oct, 196(10), 698 - 702
{Bacteremia caused by Pseudomonas aeruginosa in patients with AIDS}; Bouza Santiago E et al.; Severe infections caused by Pseudomonas aeruginosa are uncommon in patients infected with HIV . These infections are usually recurrent, bear a poor prognosis and their potential for nosocomial transmission is questioned . From May 1991 to December 1994, a total of 2,739 admissions were recorded at the VIH Unit in our hospital . Seven of those patients suffered 9 episodes of P . aeruginosa bacteremia (3.28 episodes/1,000 admissions) . All cases were acquired nosocomially . The mean CD4 count was 30.7 cells/ml . Mortality associated with Pseudomonas aeruginosa bacteremia was 43% . With molecular typing techniques homologies over 98% were demonstrated for the two episodes in patient 4 and the episode in patient 3 (very close in time, 1993) and almost 99% between the isolate from patient 7 and the two isolates (identical) from patient 6 . The other three isolates had relationship (to themselves and to the other isolates) lower than 97% . Our findings suggest that patient-to-patient transmission of severe infections caused by P . aeruginosa in patients with AIDS.

Rev Clin Esp, 1996 Oct, 196(10), 692 - 7
{The spectrum of bronchopulmonary infection caused by Pseudomonas aeruginosa in patients infected with the human immunodeficiency virus}; Santin Cerezales M et al.; OBJECTIVE: To describe the clinical spectrum of bronchopulmonary infections caused by Pseudomonas aeruginosa in patients infected with HIV . METHODS: A retrospective study of cases with P . aeruginosa pneumonia and tracheo-bronchitis in patients infected with HIV, diagnosed over a 5-year period (January 1990-December 1994) in a third level university hospital . RESULTS: Seven patients with pneumonia and 15 with tracheobronchitis were identified, with a mean age of 33 years . All patients were in an advanced stage of immunosuppression (median CD4 count 11/mm3) and 21 (95%) had AIDS . In 6 cases (86%) pneumonia was acquired in the community and no patient had severe neutropenia . Clinical presentation ranged from severe pneumonia with respiratory insufficiency and shock to subacute less severe disease . Two patients (29%) died in the first episode as a result of pneumonia . The combination of cough, purulent expectoration and fever was the usual presenting form of tracheo-bronchitis . Nine out of the 15 patients (60%) had been treated previously because of tracheo-bronchitis and/or bacterial pneumonia episodes not caused by P . aeruginosa . Twelve patients required hospital admission; ten patients (83%) because of tracheo-bronchitis symptoms . The initial response to therapy was satisfactory, but 12 (80%) relapsed, with 2.6 relapses per patient after a mean follow-up of 7.4 months . CONCLUSIONS: P . aeruginosa bronchopulmonary infections emerge in late stages of HIV disease . P . aeruginosa should be considered in the differential diagnosis of every patient with pneumonia and advanced AIDS, even in the absence of the traditionally reported risk factors . Initially, tracheo-bronchitis responds well to therapy, but its management is difficult because of the frequent relapses and the development of antibiotic resistance.

Rev Clin Esp, 1996 Oct, 196(10), 684 - 91
{Pseudomonas aeruginosa bacteremia in patients with HIV infection: clinicoepidemiologic study of 17 episodes}; Salavert Lleti S et al.; BACKGROUND: Bacteremic infection with Pseudomonas aeruginosa is an uncommon and late phenomenon in the natural history of infection with human immunodeficiency virus (HIV) . Our objective was to study the clinico-epidemiological characteristics of P . aeruginosa bacteremia (PAB) in patients infected with HIV . PATIENTS AND METHODS: A retrospective study of 17 episodes of PAB in 16 patients infected with HIV in three tertiary hospitals in the Valencia Community . Data were collected by means of a protocol designed to obtain clinical and epidemiological information . RESULTS: Fourteen out of 16 HIV-positive patients with PAB were males and in nine patients the risk factor for the acquisition of HIV was parenteral drug abuse . Eighty-one percent (13 patients) met diagnostic criteria for AIDS . Fourteen patients had less than 100 CD4 lymphocytes/mm3 at diagnosis of bacteremia (mean value 25.8) . PAB was acquired in the community in 13 episodes (76.4%) . Nine patients (56.2%) had received some type of antimicrobial therapy during the last month, 12 (75%) were taking anti-retroviral therapy, and 6 (37.5%) were receiving prophylaxis against P . carinii . The most frequent source for PAB was pneumonia with 7 episodes (41.2%), followed by the intravascular catheter infection with 2 (12%) . In six episodes (35.3%) no source was identified . Only one episode was evaluated as recurrence . Only two of the seven patients with pneumonia had pulmonary cavitation in the chest X-ray . Fifteen episodes resolved (94%) and in only one case was dead directly related to PAB . CONCLUSIONS: In our experience, PAB in patients infected with HIV emerges in the advanced stages of disease . The most common source was the lower respiratory tract and in most cases PAB was acquired in the community . The mortality rate resulting from PAB was lower than that previously reported in the literature.

Rev Clin Esp, 1996 Oct, 196(10), 678 - 83
{Respiratory infection caused by Pseudomonas aeruginosa in patients with HIV infection}; Arrizabalaga Aguirreazaldegui J et al.; OBJECTIVE: Descriptive study of severe respiratory infections caused by Pseudomonas aeruginosa in patients with HIV infection . METHODS: Review of clinical records of HIV-positive patients admitted in a tertiary hospital from 1-1-1990 to 9-30-1995 . The patients included in this study were those with P . aeruginosa recovered from respiratory or blood samples and respiratory symptoms . RESULTS: Forty patients suffered 77 episodes . The incidence in the HIV-positive admitted population during the study period was 2.6% but 3.2% in the last year . Ninety-seven percent of patients met criteria for AIDS . Twenty-eight patients (70%) had other bacterial infections . In 78% of patients no classical predisposing factor was found for this infection . Twenty-seven patients (67.5%) came regularly to the day hospital or had been previously admitted to the hospital (recent hospitalization) . Twenty-five episodes (32.5%) were tracheo-bronchitis and 50 pneumonias (65%), of which 14 (28%) were cavitated and 7 (14%) bacteremic . Forty-nine percent of episodes were recurrences . Ten out of 15 tracheo-bronchitis in the first episode recurred; the new episode was more severe in 7 patients . Five patients received secondary prophylaxis after the second episode (4 with tobramycin in aerosol and 1 with colistin in aerosol) . Two patients had recurrences after 86 and 62 days (mean: 74; SD: 12) . Eight out of the 11 patients who did not receive prophylaxis (73%) had recurrence after a mean of 23 days (SD: 10) (p = 0.002) . The mortality rate associated with P . aeruginosa was 22.5% . CONCLUSIONS: The incidence of P . aeruginosa infections in our HIV-positive patients was 2.6% (3.2% in the last year) . It is associated with severe immunosuppression and previous bacterial infection . The subacute involvement of the lower respiratory tract is most common . Over 50% of infections tend to recur more severely . The secondary prophylaxis increased the symptom-free period, although the number of patients who received it was small.

Nippon Saikingaku Zasshi, 1996 Oct, 51(4), 1049 - 53
{An improved phagocytic plaque method using gram-negative bacilli}; Tomiya Y et al.; The phagocytic plaque method developed for evaluation of the phagocytic function of leukocytes is not only very useful but convenient for both basic and clinical investigations . So far, only Staphylococcus aureus has been demonstrated to give clear phagocytic plaques . In this study, we successfully obtained clear phagocytic plaques with gram-negative bacilli (Pseudomonas aeruginosa and Escherichia coli) . The plaques given by E . coli were morphologically nearly the same as those by S . aureus, whereas those of P . aeruginosa were elongated.

Thorax, 1996 Oct, 51(10), 1023 - 7
Long-term survival and nutritional data in patients with cystic fibrosis treated in a Danish centre; Nir M et al.; BACKGROUND: Adequate nutrition and optimal treatment of bronchopulmonary infections are both of critical importance in maintaining the health of patients with cystic fibrosis . The cystic fibrosis centre in Copenhagen has followed a regimen of very early and aggressive antimicrobial treatment, especially against Pseudomonas aeruginosa infection . An unrestricted diet of low fat and high protein without hyperalimentation was recommended before 1985 which was then changed to a high fat, high calorie intake . METHODS: The overall impact of the treatment regimen was evaluated by a cross sectional analysis of all 223 patients who attended the centre in 1989 . Growth and nutritional parameters were combined with lung function parameters and with a retrospective analysis of chronic P aeruginosa infection and its duration . Survival curves for all 313 patients treated at the centre since 1949 were calculated . RESULTS: All the patients with cystic fibrosis had normal height, although the final height was achieved a little later than in healthy controls . Body weight was lower than normal in males above 15 and in females above 10 years of age . The body mass index (BMI), which was approximately 98% of normal in the younger patients, declined to 90% in adult men and to 83% in adult women with cystic fibrosis, and was strongly correlated with lung function parameters . In 1989 the median age of survival of all patients treated in the centre since 1949 was 30 years (32 years in males and 29 years in females) . CONCLUSIONS: The overall treatment regimen in the cystic fibrosis centre in Copenhagen is associated with growth and survival rates that are at least equal to those in other cystic fibrosis centres in other countries.

J Antimicrob Chemother, 1996 Oct, 38(4), 707 - 11
Cefepime activity against Pseudomonas aeruginosa: evaluation of Etest and two disc diffusion methods; Stes P et al.; The in-vitro susceptibility 244 consecutive, non-duplicate clinical isolates of Pseudomonas aeruginosa cefepime and ceftazidime was studied . Using an agar dilution method to measure susceptibility, the antibiotics showed comparable activity . The Etest and two types of agar diffusion discs were compared with the reference agar dilution method, and showed an absolute interpretive agreement value of over 95%.

Mol Microbiol, 1996 Oct, 22(2), 239 - 54
Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa; Raivio TL et al.; RegA is a transcriptional activator that controls exotoxin A (ETA) production in Pseudomonas aeruginosa . To date, functional assays performed with the purified protein have not clearly defined the molecular mechanism of action of RegA . In this study, we sought to identify important coding regions of regA by analysing the sequences around linker insertion mutations in regA that affected toxA transcription . First, we constructed a strain with the regAB locus deleted from the chromosome, PA103 delta regAB::Gm . toxA transcription was obliterated in strain PA103 delta regAB::Gm, demonstrating that the regAB locus is essential for ETA production . Next, we constructed a series of 6 bp linker insertion mutations distributed throughout regA . These regA linker insertion mutants were sequenced and screened in PA103 delta regAB::Gm for their effects on regulation of ETA production . Six linker insertion mutations occurring between amino acids (aa) 53 and 163 of RegA were isolated that resulted in depression of toxA transcription to varying levels relative to the parental regAB locus . One of these linker insertion mutations (pTR53), resulted in a lack of iron-regulated ETA production and occurred directly upstream from a predicted transmembrane alpha-helix . The other five linker mutations (pTR88, pTR124, pTR132, pTR132-2 and pTR163) occurred within or flanked a region of RegA between aa 87-142 with similarity to the transcriptional activation domains of ToxR, VirG and OmpR . These data suggest the presence of a previously unidentified transcriptional activation domain in RegA between aa 87-142 and implicate the predicted transmembrane alpha-helix in the N-terminus as being involved in sensory transduction.

Cathet Cardiovasc Diagn, 1996 Oct, 39(2), 168 - 70; discussion 171
Fatal infection of coronary stent implantation; Leroy O et al.; Among systemic infections occurring after percutaneous transluminal coronary angioplasty (PTCA) and coronary stent implantation, septic cardiac complications are rare . We report a new case of infective aneurysm of the left anterior descending coronary artery (LAD) following stent implantation . Infective mitral endocarditis due to Pseudomonas aeruginosa occurring a few weeks after stenting led to search for stent infection . Coronary angiography revealed a saccular aneurysm of the LAD . Despite surgical repair, a fatal outcome resulted.

J Am Soc Nephrol, 1996 Oct, 7(10), 2183 - 91
Transmembrane passage of cytokine-inducing bacterial products across new and reprocessed polysulfone dialyzers; Sundaram S et al.; The widespread use of bicarbonate dialysate and reprocessed high-efficiency and "high-flux" dialyzers has raised concerns about the increased risk of reverse-transfer of dialysate contaminants into the blood compartment . To evaluate this concern, the reverse-transfer of bacterial products from contaminated bicarbonate dialysate into the blood compartment was compared during in vitro dialysis with new or reprocessed high-flux polysulfone dialyzers . In vitro dialysis was carried out at 37 degrees C by use of a counter-current recirculating loop dialysis circuit with either new high-flux polysulfone dialyzers or dialyzers reprocessed once or 20 times with formaldehyde (0.75%) and bleach (< 1%) with an automated system . Heparinized whole blood from healthy volunteers was circulated through the blood compartment, and bicarbonate dialysate was circulated in the dialysate compartment . The dialysate was challenged sequentially by 1:1000 and 1:100 dilutions of a sterile Pseudomonas aeruginosa culture supernatant (bacterial challenge) . Samples were drawn from the blood and dialysate compartments 1 h after each challenge . Peripheral blood mononuclear cells (PBMC) were harvested by Ficoll-Hypaque separation from whole blood in the blood compartment and a 5 x 10(6) PBMC/mL cell suspension was prepared . Likewise, dialysate samples (0.5 mL) were added to 0.5 mL suspension of 5 x 10(6) PBMC/mL drawn at baseline . All PBMC suspensions were incubated upright in a humidified atmosphere at 37 degrees C with 5% CO2 for 24 h, and total interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF alpha) cytokine production (cell-associated and secreted) was measured by radioimmunoassay . Eight experiments were performed for each arm of the study with the same donor for each arm . One hour after contaminating the dialysate with a 1:1000 dilution of the bacterial challenge, IL-1 alpha production by PBMC harvested from the blood compartment was 160 +/- 0, 171 +/- 11, and 270 +/- 35 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.004) . One hour after challenging the dialysate with 1:100 dilution, IL-1 alpha production by PBMC harvested from the blood compartment was 188 +/- 20, 228 +/- 35, and 427 +/- 67 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.006) . IL-1 alpha production by PBMC from dialyzers reprocessed 20 times was significantly greater than both new and dialyzers reprocessed once . However, there were no significant differences between new dialyzers and dialyzers reprocessed once . Similarly, after the 1:1000 challenge, TNF alpha production by PBMC harvested from the blood compartment was 160 +/- 0, 160 +/- 0, and 213 +/- 22 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.008) . After the 1:100 challenge, TNF alpha production was 168 +/- 8, 188 +/- 20, and 225 +/- 32 pg, respectively, for new dialyzers, dialyzers reprocessed once, and dialyzers reprocessed 20 times (P = 0.20) . These results demonstrate that reprocessing of high-flux polysulfone dialyzers with bleach increases the risk of reverse-transfer of bacterial products from contaminated dialysate, and this risk appears to increase with the number of reuses . Consequently, units that reprocess membranes with bleach and have suboptimal water quality might subject their patients to a higher risk of cytokine-related morbidity.

J Cataract Refract Surg, 1996 Oct, 22(8), 1116 - 20
Acute endophthalmitis outbreak after cataract surgery; Arsan AK et al.; PURPOSE: To evaluate the source of organisms causing an epidemic of postoperative endophthalmitis and to emphasize the importance of prompt intervention with an early diagnosis . SETTING: S.B . Ankara Hospital Eye Department, Ankara, Turkey . METHODS: Thirteen patients who had surgery on the same day and developed acute postoperative endophthalmitis were evaluated . Clinical patterns were observed and intraocular cultures and stains performed in 10 eyes . Broad-spectrum intravitreal antibiotics were injected on an empirical basis . RESULTS: Intravitreal cultures showed Pseudomonas aeruginosa in four cases and coagulase-negative staphylococci in three cases; three cases were culture negative . P . aeruginosa were also isolated from irrigation solutions used on the same day . Two patients with P . aeruginosa had a visual acuity of 20/200 and 20/300, respectively . CONCLUSION: The different culture results were probably related to the amount of inoculation, individual risk factors, and the subconjunctival antibiotic injection given at the end of surgery . That one patient with P . aeruginosa endophthalmitis retained a visual acuity of 20/200 shows the importance of rapid intravitreal antibiotic treatment.

J Pharmacol Toxicol Methods, 1996 Oct, 36(2), 73 - 6
A simple in vitro model to test the efficacy of antimicrobial agents released from dressings; Grzybowski J et al.; A new model for in vitro evaluation of an activity of antibacterial agents released from delivery systems containing antibiotics or antiseptics was developed . The model was composed of two layers of agar gel of two different concentrations in PBS . In the upper gel, the wells were cut and filled with polyurethane sponges saturated with bacterial broth culture . The sponges were then covered with tested dressings containing antibacterial agent . The model was tested by using two bacterial strains, Pseudomonas aeruginosa or Staphylococcus aureus and experimental collagen dressing with doxycyclin, amikacin, or with silver sulfadiazine . The number of colony-forming units (CFU) in the polyurethane sponges was significantly lower under the dressings with antibacterial agents as compared to the control (the same dressing without antimicrobials) during 3 days of observation . The new in vitro model can be recommended for examination of different antibacterial delivery systems instead of experimentally infected wounds in laboratory animals or instead of the other in vitro models.

Eur Respir J, 1996 Oct, 9(10), 2145 - 50
Pseudomonas aeruginosa adherence to remodelling respiratory epithelium; de Bentzmann S et al.; Pseudomonas aeruginosa is an opportunistic organism, which frequently colonizes the respiratory tract of patients with impaired host defence . In cystic fibrosis (CF) patients, this pathogen causes a progressive destructive bronchitis and bronchiolitis and is responsible for high mortality . Normal respiratory epithelium is protected against bacteria via mucus and mucociliary clearance . Alteration of mucociliary clearance and of glycosylation of mucins in CF facilitates the access of bacteria to the underlying airway epithelial cells . Intact respiratory epithelium does not bind P . aeruginosa, whereas injured respiratory epithelium is highly susceptible to P . aeruginosa adherence . We found that the high affinity of respiratory epithelium, from CF and non-CF sources, for P . aeruginosa, during the wound repair process is related to the apical expression of asialo ganglioside M1 (aGM1) . The affinity of repairing respiratory epithelium for P . aeruginosa is time-dependent, and is related to transient apical expression of aGM1 at the surface of repairing respiratory epithelial cells . CF respiratory epithelial cells apically express more aGM1 residues with relation to an increased affinity for P . aeruginosa than non CF cells . High epithelial damage followed by repair represents a major cause of P . aeruginosa adherence to airway epithelium in cystic fibrosis . However, P . aerurignosa adherence and colonization are not restricted to cystic fibrosis disease and P . aeruginosa pneumonia may also occur in severely immunocompromised patients, suggesting that epithelial injury and decreased host-response favour the colonization of the airways by P . aeruginosa.

Mol Microbiol, 1996 Oct, 22(1), 97 - 108
Identification and characterization of AlgZ, an AlgT-dependent DNA-binding protein required for Pseudomonas aeruginosa algD transcription; Baynham PJ et al.; Transcriptional activation of the Pseudomonas aeruginosa algD gene results in high-level synthesis of the capsular polysaccharide alginate, an important P . aeruginosa virulence factor expressed in cystic fibrosis (CF) patients with chronic pulmonary disease . In this study, electrophoretic mobility-shift assays were used to identify a novel protein (AlgZ), which binds specifically to a sequence located 280 bp upstream of the algD promoter . While AlgZ-binding activity did not require the response regulators AlgB or AlgR, expression of AlgZ was found to be absolutely dependent on the alternative sigma factor AlgT . Electrophoretic mobility-shift assays and copper-phenanthroline footprinting localized AlgZ binding to a 36 bp algD region, which includes several helical repeats . A collection of alginate-producing (mucoid) and non-mucoid P . aeruginosa strains, derived from CF patients, was characterized for AlgZ-binding activity . In all cases, AlgZ binding to algD sequences was observed when extracts derived from mucoid P . aeruginosa CF isolates were examined . However, this binding activity was not present when extracts from non-mucoid P . aeruginosa CF isolates were tested . Oligonucleotide mutagenesis was employed to create an algD allele with a 4 bp mutation in the predicted AlgZ-binding site (algD38) and a heterologous substitution allele (algD40), in which the entire AlgZ-binding site was replaced with a non-specific DNA sequence of identical size . When the algD38 mutation was cloned into an algD-cat transcriptional fusion, this resulted in a 28-fold reduction in algD expression, whereas the algD40 mutation abolished algD transcription, indicating that AlgZ acts as an activator of algD transcription . These results support the hypothesis that activation of algD involves the formation of a high-order looped structure allowing for multivalent contacts between AlgZ, AlgR and RNA polymerase containing the alternative sigma factor AlgT . Characterization of the molecular details of algD activation will provide insights into the control of other prokaryotic and eukaryotic promoters that utilize multiple activators.

Biochem Mol Biol Int, 1996 Oct, 40(2), 403 - 8
Binding between Pseudomonas aeruginosa adhesins and human salivary, tracheobronchial and nasopharyngeal mucins; Reddy MS; Endogenous aspiration of nasooropharyngeal secretions is a major source of Pseudomonas aeruginosa to lungs . To understand the mechanism of binding between P . aeruginosa and nasooropharyngeal secretions, we have utilized an overlay binding assay employing P . aeruginosa nonpilus protein adhesins (18 kDa and 15 kDa) and mucins from human saliva, tracheobronchial and nasopharyngeal secretions . Nasopharyngeal mucin bound to both the 18 kDa and 15 kDa adhesins . A high molecular weight salivary mucin and tracheobronchial mucin bound only to the 18 kDa adhesin . On the other hand, asialonasopharyngeal and asialotracheobronchial mucins did not bind to either of the adhesins . Binding of nasopharyngeal mucin to the 18 kDa and 15 kDa was not inhibitable by glycoproteins, bovine submaxillary mucins and sialic acid . Further, a low molecular weight salivary mucin which contains sialylated oligosaccharides also did not bind to either of the adhesins . Collectively, the results suggest that binding between P . aeruginosa and mucins may involve specific receptors and adhesins.

Antimicrob Agents Chemother, 1996 Oct, 40(10), 2410 - 2
Differential release of smooth-type lipopolysaccharide from Pseudomonas aeruginosa treated with carbapenem antibiotics and its relation to production of tumor necrosis factor alpha and nitric oxide; Yokochi T et al.; Endotoxin release from Pseudomonas aeruginosa treated with cell wall-active carbapenem antibiotics and its effect on the production of tumor necrosis factor alpha and nitric oxide were examined . Treatment of bacteria with imipenem induced much lower levels of endotoxin release than treatment with meropenem . The endotoxin released was demonstrated to be of the smooth type and O-specific polysaccharide-rich . The exposure of the filtrates of P . aeruginosa treated with imipenem to physiologically relevant cells caused low-level production of tumor necrosis factor alpha and nitric oxide, while similar treatment with meropenem induced high levels of production.

Antimicrob Agents Chemother, 1996 Oct, 40(10), 2280 - 4
Evaluation of susceptibility of gram-positive and -negative bacteria to human defensins by using radial diffusion assay; Takemura H et al.; Defensins are small cationic bactericidal peptides present abundantly in the granules of polymorphonuclear neutrophils (PMNs) . Human PMNs contain four defensins termed HNP-1 to HNP-4 . We used a new assay system in agar plates, the radial diffusion assay, to evaluate the effects of human defensins against gram-positive and -negative bacteria . A crude mixture of HNP-1, -2, and -3 (crude HNPs) was purified from human PMN extracts by reversed-phase high-pressure liquid chromatography (RP-HPLC) . The different components were later separated by RP-HPLC and gel permeation chromatography . We compared the antibacterial activities of purified HNP-1, -2, and -3 against Escherichia coli, Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus, and methicillin-resistant S . aureus strains using the radial diffusion assay . The antibacterial activities of HNP-1 and HNP-2 against all strains tested were similar to those of the crude HNPs, but the activity of HNP-3 was less than those of the other defensins . To quantitate the activities of HNPs against different bacteria, we defined the minimal dose of crude HNPs forming a detectable clear zone around the bacteria as the minimal inhibitory dose (MID) and determined the MIDs for 10 strains of E . coli, 12 strains of P . aeruginosa, 10 strains of methicillin-susceptible S . aureus, and 12 strains of methicillin-resistant S . aureus isolates, including clinical isolates . In general, the MIDs of the HNPs were similar against similar bacterial species . However, the MIDs for P . aeruginosa were higher than those for the other organisms tested . The radial diffusion assay is suitable as a screening test for measuring the susceptibilities of isolates to defensins, because it is sensitive and simple and has good reproducibility.

Antimicrob Agents Chemother, 1996 Oct, 40(10), 2271 - 5
Direct evidence for antipseudomonal activity of macrolides: exposure-dependent bactericidal activity and inhibition of protein synthesis by erythromycin, clarithromycin, and azithromycin; Tateda K et al.; Several previous investigators have reported that long-term administration of certain macrolides is efficacious in patients with persistent pulmonary Pseudomonas aeruginosa infections, even though the clinically achievable concentrations of these medications are far below their MICs . In the present study, we examined how sub-MICs of macrolide antibiotics affect the viability of and protein synthesis in several strains of P . aeruginosa . We report that 48 h, but not 12 or 24 h, of growth on agar containing a clinically achievable concentration of azithromycin (0.5 microgram/ml, 1/128 the MIC) significantly reduces the viability of strain PAO-1 . Similar effects were seen with erythromycin and clarithromycin at 2 micrograms/ml (1/128 and 1/64 the respective MICs), whereas josamycin, oleandomycin, ceftazidime, tobramycin, minocycline, and ofloxacin had no effect on viability, even following 48 h of incubation with concentrations representing relatively high fractions of their MICs . The bactericidal activity of azithromycin seen following 48 h of incubation was not limited to strain PAO-1 but was also seen against 13 of 14 clinical isolates, including both mucoid and nonmucoid strains . Although viability was not decreased prior to 48 h, we found that 4 micrograms of azithromycin per ml inhibits protein synthesis after as little as 12 h and that protein synthesis continues to decrease in a time-dependent manner . We likewise found that P . aeruginosa accumulates azithromycin intracellulary over the period from 12 to 36 h . These results suggested that sub-MICs of certain macrolides are bactericidal to P . aeruginosa when the bacteria are exposed to these antibiotics for longer periods . Exposure-dependent intracellular accumulation of the antibiotic and inhibition of protein synthesis may partially account for the antipseudomonal activity of macrolides over relatively prolonged incubation periods.

Biophys J, 1996 Oct, 71(4), 2138 - 43
Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence; Hansen JE et al.; Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue . Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water . In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein . The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin . Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue . However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue . The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid . Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein.

Microbiology, 1996 Oct, 142 ( Pt 10), 2785 - 93
Isolation of an IHF-deficient mutant of a Pseudomonas aeruginosa mucoid isolate and evaluation of the role of IHF in algD gene expression; Delic-Attree I et al.; The role of integration host factor (IHF) in the regulation of alginate synthesis was investigated in a mucoid strain of Pseudomonas aeruginosa (strain CHA) isolated from a cystic fibrosis patient . Escherichia coli strain BL21(DE3) was made IHF-deficient by inactivation of its chromosomal IHF genes, himA and himD, then used as host strain to overproduce P . aeruginosa IHF . The purified recombinant IHF protein was used to determine the affinity of IHF for the two IHF binding sites in the algD promoter . The Kd values were determined to be 130 nM for algD IHF site 2 and about 2 microM for algD IHF site 1 . Two IHF-deficient mutants of P . aeruginosa strain CHA were constructed by insertional inactivation of the himA gene, and the activity of the algD promoter was determined using transcriptional fusion with xylE as reporter gene . The expression of algD, the structural gene for GDP-mannose dehydrogenase, was decreased three- to fourfold in the himA mutants under conditions of high salinity and nitrogen limitation . Assays of alginate production by cultures grown on agar plates indicated that the IHF-deficient mutants synthesized 50% less polymer than the mucoid parental strain . These results demonstrate clearly that although IHF is dispensable for alginate production, himA expression is required for full activation of algD expression.

J Biomed Mater Res, 1996 Oct, 32(2), 271 - 8
Measurement of bacterial growth rates on polymers; Barton AJ et al.; A video microscope system and a mathematical model were developed to observe and model the early stage of bacterial growth on polymer surfaces . Glass slides were coated with polyorthoester, poly(L-lactic acid), and polysulfone, and inserted into a laminar flow cell to expose them to bacterial cultures of Staphylococcus epidermidis, Pseudomonas aeruginosa, or Escherichia coli . The free energy of adhesion (delta Fadh) was determined from contact-angle measurements . The microscopic observations along with the mathematical model allowed measurement of the rates of adhesion, release, and growth . The growth rate of P . aeruginosa on the various surfaces correlated to the delta Fadh . The growth rates of all species on all of the surfaces were slower than the growth rates of the bacteria in suspension . The mathematical model is valid for early growth before the bacteria form a complete monolayer, and is useful in predicting and modeling early growth of bacteria on implanted biomaterials.

J Clin Microbiol, 1996 Oct, 34(10), 2386 - 90
DNA fingerprinting by infrequent-restriction-site amplification; Mazurek GH et al.; Identification of bacterial strains by DNA fingerprinting facilitates epidemiologic studies and improves disease control . For some species of organisms, no typing method is available; for others, typing methods are tedious . We developed a method of amplifying DNA sequences flanking infrequent restriction sites by PCR and used the method to produce strain-specific electrophoretic patterns from crude bacterial lysates . This method of fingerprinting is rapid, sensitive, and widely applicable . Identical enzymes, adaptors, primers, and PCR conditions were used to characterize 32 Mycobacterium avium-M . intracellulare isolates, 4 Pseudomonas aeruginosa isolates, and 4 Staphylococcus aureus isolates.

Am J Respir Crit Care Med, 1996 Oct, 154(4 Pt 2), S183 - 6
Differential gene expression by Pseudomonas aeruginosa during interaction with respiratory mucus; Lory S et al.; Pseudomonas aeruginosa is a common respiratory tract pathogen that causes serious infections in patients with cystic fibrosis . A number of putative virulence factors have been characterized in several laboratories, and some have been implicated in human infections, based on criteria such as the phenotype of isolates from infected patients, an immune response to a particular antigenic factor, and the effect of a virulence factor on infectivity in an animal model . We have developed a series of genetic tools to study the selective regulation of expression of P . aeruginosa genes during interactions of the pathogen with host tissues . These tools are based on direct enrichment of bacteria, when a particular promoter is induced or repressed . We have found that interaction of bacteria with mucus from patients with cystic fibrosis results in marked induction of expression of several genes, including one that encodes a lipopolysaccharide biosynthetic enzyme, a gene for a protein responsible for uptake of the ferric pyochelin siderophore, and a new gene homologous with a class of iron-responsive repressors . The tools described here are useful for identification of induced or repressed genes in various animal models of infection or in controlled laboratory conditions that mimic natural infections of humans . Such genes might not be detectable when bacteria are cultured in laboratory conditions, and these tools are therefore useful for general probing of a bacterial genome for genes regulated during different stages of infection.

Am J Respir Crit Care Med, 1996 Oct, 154(4 Pt 2), S175 - 82
How mutant CFTR may contribute to Pseudomonas aeruginosa infection in cystic fibrosis; Pier GB et al.; Patients with cystic fibrosis (CF) have a pronounced hypersusceptibility (80 to 90%) to Pseudomonas aeruginosa infection . We hypothesized that airway epithelial cell ingestion of bacteria followed by cellular desquamation may protect the lung from infection, and epithelial cells expressing mutant forms of the cystic fibrosis transmembrane conductance regulator (CFTR) may be defective in this function . We found that transformed human airway epithelial cells homozygous for the delta F508 allele of CFTR were significantly defective in uptake of P . aeruginosa compared with the same cell line complemented with the wild-type allele of CFTR . Partial membrane expression of the delta F508 CFTR protein occurs in cells grown at 26 degrees C, and under these conditions uptake of P . aeruginosa occurred at levels comparable to cells with a wild-type allele of CFTR . Epithelial cell ingestion assays using isogenic bacterial strains differing in lipopolysaccharide (LPS) phenotype, along with inhibition studies, identified the LPS-core oligosaccharide as the bacterial ligand for epithelial cell invasion . Inhibition of epithelial cell ingestion of P . aeruginosa in a neonatal mouse lung infection model led to increased levels of bacteria in the lungs 24 and 48 h after infection . Defective epithelial cell internalization of P . aeruginosa may be a critical factor in hypersusceptibility of CF patients to chronic lung infections.

Am J Respir Crit Care Med, 1996 Oct, 154(4 Pt 2), S170 - 4
Recognition of mucin by the adhesin-flagellar system of Pseudomonas aeruginosa; Ramphal R et al.; Pseudomonas aeruginosa colonizes the mucus of patients with chronic lung diseases by a specific mechanism involving an adhesin-receptor system . Several adhesins have been implicated in the adhesion of P . aeruginosa to cells, but the identity of the principal adhesin(s) involved in adhesion to mucin is unknown . Mutagenesis studies have indicated that P . aeruginosa adhesion is under the control of the rpoN gene, which also regulates pilin synthesis, flagellum formation, and other functions . Mutagenesis of certain flagellar genes that are not controlled by RpoN, e.g., flif, also indicates a close relationship between adhesion and flagellar genes and not necessarily an independent effect of rpoN on adhesion . Mutants of certain early flagellar genes lead to the loss of both adhesion and motility, whereas mutants of certain late genes, e.g., fliC, the gene for flagellin, lose motility but retain adhesion . Recent studies indicate that both motility and adhesion are regulated by a two-component regulatory system called fleS-R, which in turn is controlled by another regulator in a cascade that involves rpoN . A fleR mutant possessing pili adheres poorly to mucins, definitively showing that pili do not play a major role in adhesion to mucin . It is unclear whether the adhesin is a flagellar protein or another protein that uses the flagellar export apparatus for localization or both . Finding the gene under control of rpoN may provide answers to these questions.

Am J Respir Crit Care Med, 1996 Oct, 154(4 Pt 2), S163 - 9
Interactions between glycoconjugates from human respiratory airways and Pseudomonas aeruginosa; Scharfman A et al.; Pseudomonas aeruginosa binds to different glycoconjugates in vitro . As six other bacteria, it binds to several glycolipids, mainly asialo GM1 and asialo GM2 . Asialo GM1 has been reported to exist at the surface of cystic fibrosis cells . The binding of P . aeruginosa to asialo GM1 involves the pili, especially the C-terminal part of pilin that recognizes the GaINAc(beta 1,4) Gal sequence of asialo GM1.P . aeruginosa may also bind to sialylated membrane-bound glycoproteins . Human salivary and respiratory mucins are also recognized by P . aeruginosa . Mucins represent the main components of mucus . The peptide part (apomucin) of this broad family of secreted glycoproteins is encoded by several mucin genes . The apomucins are covered by a large number of carbohydrate chains that can be remarkably different and represent a mosaic of sites for attachment of microorganisms . The binding of P . aeruginosa to mucins involves outer membrane proteins and mucin carbohydrate chains that are structurally different from the carbohydrate recognized by pillin . Airway and salivary mucins secreted by patients suffering from cystic fibrosis (CF) show alterations in their carbohydrate moiety . The increased sulfation of airway mucins seems to correspond to a primary defect . Other abnormalities such as increased sialylation or fucosylation have also been detected . The binding of P . aeruginosa to airway or salivary mucins is increased in CF . However, the precise link between the carbohydrate alterations and the increased binding of P . aeruginosa to CF mucins remains to be elucidated.

Am J Respir Crit Care Med, 1996 Oct, 154(4 Pt 2), S155 - 62
Receptors in the Pseudomonas aeruginosa adherence to injured and repairing airway epithelium; de Bentzmann S et al.; In the normal respiratory tract, the airway epithelial surface is protected from pathogenic bacterial colonization by the mucociliary clearance . The mucins present in the gel mucus layer exhibit a high diversity of carbohydrate receptors that allow specific bacterial recognition followed by bacterial and mucus elimination . As soon as the mucociliary clearance mechanism is impaired, the bacterial attachment to mucins in association with mucus stasis represent critical pathways for bacterial colonization of the airway epithelium . Several sources of injury may damage the epithelial integrity and induce partial or complete epithelial shedding, exposing cellular receptors and unmasked extracellular matrix (ECM) components that can be recognized by bacterial adhesins . Laminin and type I and IV collagens represent sites of Pseudomonas aeruginosa attachment to the ECM components . During airway epithelium repair after injury, particularly in cystic fibrosis (CF), the repairing cells exhibit apical receptors such as asialylated gangliosides (asialo GM1) to which P . aeruginosa adheres . The identification of the different receptors for P . aeruginosa, present either on the ECM proteins or on the apical surface of the remodeled airway epithelium, particularly in repairing respiratory CF epithelial cells, is a prerequisite to further therapeutic strategies to prevent airway colonization by P . aeruginosa.

Anesthesiology, 1996 Oct, 85(4), 883 - 8
Epidural catheter reconnection . Safe and unsafe practice; Langevin PB et al.; BACKGROUND: An in vitro model of epidural catheter contamination was used to determine if disconnected catheters can be safely reconnected . METHODS: Epidural catheters were filled with brain-heart infusion (BHI) broth or preservative-free saline containing 5 micrograms/ml fentanyl . Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus (1.10(5) colony-forming units) was injected into the initial 1.1 +/- 0.24 inch (2.75 +/- 0.60 cm) of the catheters . To study the effect of bacteria settling in a vertically oriented catheter on the advancement of bacteria along the catheter, bacteria were incubated with catheters in the vertical and the horizontal positions . To determine if bacteria are swept further into a catheter when fluid in it is displaced, catheters were inclined 30 degrees and the fluid in them was allowed to drain from the distal end to various extents . Bacteria were incubated with the catheter held horizontally . After incubation, the catheters were serially sectioned, and the resulting segments were eluted with buffered saline-containing gelatin (BSG), which was collected on BHI agar plates for colony counts . This determined if a segment of the catheter remained internally sterile distal to the point of disconnection . Effectiveness of decontaminating the exterior of the catheter was also tested as follows: Catheters (n = 10) were first immersed in BSG containing 1.10(5) S, aureus, immediately immersed in betadine for 2 min, exposed to air for 3 min, cut with a sterile instrument, and reconnected to a sterile screw cap catheter connector . Reconnected catheters were perfused with 10 ml BSG for 1 hr . Collected perfusate (100 microliters) was removed for direct colony count; the remaining perfusate was mixed with an equal volume of BHI and incubated overnight . A 100 microliters aliquot of BHI-BSG mixture was sampled the next day . No bacteria were cultured from either the perfusate or BHI-BSG mixture . RESULTS: Eight hours after contamination, as long as the fluid in the catheter was static, no bacteria were detected more than 13 inches (32.5 cm) from the contaminated end of catheters filled with BHI and no more than 8 inches (20 cm) from the end of those filled with fentanyl solution . This finding was not affected by incubation of the catheter in the vertical position . Fluid displacement less than 8 inches (20 cm) had no effect on dissemination, but when fluid was displaced 13 inches (32.5 cm), bacteria were found at the end of the catheter, 35 inches (87.5 cm) away . No bacteria were recovered from the perfusate of reconnected catheters after the catheters were cleaned with betadine and cut with a sterile instrument . CONCLUSIONS: There may be an area distal to the disconnected end of an epidural catheter where its interior remains sterile for at least 8 hr . Such an area exists only when the fluid in the catheter remains static . Furthermore, the exterior of the catheter can be adequately cleaned to prevent bacteria from entering the catheter when reconnected at that point.

Otolaryngol Head Neck Surg, 1996 Oct, 115(4), 342 - 51
Inhibition of proteases in Pseudomonas otitis media in chinchillas; Cotter CS et al.; The treatment of chronic suppurative otitis media caused by Pseudomonas aeruginosa remains a challenging problem . The virulence of Pseudomonas is related to its secretion of two matrix metalloproteinases, alkaline protease and elastase . This experiment examines the effects of a synthetic inhibitor of matrix metalloproteinases GM 6001, or N-(2(R)-2(hydroxyamido carbonylmethyl)-4-methylpentanoyl)-L-tryptophane methylamide), in a chinchilla Pseudomonas otitis media model . Thirty chinchillas underwent bilateral subtotal tympanic membrane perforations . Twenty-four chinchillas underwent bilateral middle ear inoculation with P . aeruginosa . Chinchillas were divided into four groups of six animals after the establishment of otitis media . Animals in one group were controls; the other three groups received either gentamicin, GM 6001, or gentamicin plus GM 6001 into the external auditory canal three times daily for 4 weeks . Clearance of Pseudomonas infection occurred in three ears of three animals, all in gentamicin groups, with or without GM 6001 . Otorrhea (p = 0.0014) and external canal erythema (p = 0.025) were mild in the two gentamicin groups and moderate in the GM 6001 group when compared with bacterial controls . Animals in the GM 6001 group had the highest survival rate, less severe facial paralysis, and less vestibular toxicity than the gentamicin, gentamicin plus GM 6001, or control groups, although these differences were not statistically significant . This pilot study showed encouraging results for a role of ototopical protease inhibitors in the treatment of Pseudomonas otitis media.

Appl Environ Microbiol, 1996 Oct, 62(10), 3910 - 3
Genetic exchange in soil between introduced chlorobenzoate degraders and indigenous biphenyl degraders; Focht DD et al.; Pseudomonas aeruginosa JB2, a chlorobenzoate degrader, was inoculated into soil having indigenous biphenyl degraders but no identifiable 2-chlorobenzoate (2CBa) or 2,5-dichlorobenzoate (2,5DCBa) degraders . The absence of any indigenous chlorobenzoate degraders was noted by the failure to obtain enrichment cultures with the addition of 2CBa, 3CBa, or 2,5DCBa and by the failure of soil DNA to hybridize to the tfdC gene, which encodes ortho fission of chlorocatechols . In contrast, DNA extracted from inoculated soils hybridized to this probe . Bacteria able to utilize both biphenyl and 2CBa as growth substrates were absent in uninoculated soil, but their presence increased with time in the inoculated soils . This increase was related kinetically to the growth of biphenyl degraders . Pseudomonas sp . strain AW, a dominant biphenyl degrader, was selected as a possible parental strain . Eight of nine recombinant strains, chosen at random, had high phenotypic similarity (90% or more) to the inoculant; the other, strain JB2-M, had 78% similarity . Two hybrid strains, P . aeruginosa JB2-3 and Pseudomonas sp . JB2-M, were the most effective of all strains, including strain AW, in metabolizing polychlorinated biphenyls (Aroclor 1242) . Repetitive extragenic palindromic-PCR analysis of putative parental strains JB2 and AW and the two recombinant strains JB2-3 and JB2-M showed similar fragments among the recombinants and JB2 but not AW . These results indicate that the bph genes were transferred to the chlorobenzoate-degrading inoculant from indigenous biphenyl degraders.

J Bacteriol, 1996 Oct, 178(20), 6067 - 9
Secondary structure of the outer membrane proteins OmpA of Escherichia coli and OprF of Pseudomonas aeruginosa; Sugawara E et al.; When purified without the use of ionic detergents, both OmpA and OprF proteins contained nearly 20% alpha-helical structures, which disappeared completely upon the addition of sodium dodecyl sulfate . This result suggests that the proteins fold in a similar manner, with an N-terminal, membrane-spanning beta-barrel domain and a C-terminal, globular, periplasmic domain.

J Bacteriol, 1996 Oct, 178(20), 6064 - 6
A two-component response regulator, gltR, is required for glucose transport activity in Pseudomonas aeruginosa PAO1; Sage AE et al.; A 729-bp open reading frame (gltR) was identified in Pseudomonas aeruginosa PAO1 that encodes a product homologous to the two-component response regulator family of proteins . Disruption of gltR caused loss of glucose transport activity . Restoration of gltR resulted in wild-type levels of glucose transport . These findings indicate that gltR is required for expression of the glucose transport system in P . aeruginosa.

J Bacteriol, 1996 Oct, 178(20), 5995 - 6000
Functional analysis of the Pseudomonas aeruginosa autoinducer PAI; Passador L et al.; A series of structural analogs of the Pseudomonas aeruginosa autoinducer {PAI, N-3-oxo-dodecanoyl homoserine lactone} were obtained and tested for their ability to act as autoinducers in stimulating the expression of the gene for elastase (lasB) by measuring beta-galactosidase production from a lasB-lacZ gene fusion in the presence of the transcriptional activator LasR . The data suggest that the length of the acyl side chain of the autoinducer molecule is the most critical factor for activity . Replacement of the ring O by S in the homoserine lactone moiety can be tolerated . Tritium-labelled PAI ({3H}PAI) was synthesized and used to demonstrate the association of {3H}PAI with cells overexpressing LasR . The PAI analogs were also tested for their ability to compete with {3H}PAI for binding of LasR . Results from the competition assays suggest that once again the length of the acyl side chain appears to be crucial for antagonist activity . The presence of the 3-oxo moiety also plays a significant role in binding since analogs which lacked this moiety were much less effective in blocking binding of {3H}PAI . All analogs demonstrating competition with PAI in binding to LasR also exhibited the ability to activate lasB expression, suggesting that they are functional analogs of PAI.

J Bacteriol, 1996 Oct, 178(20), 5884 - 9
A new Azotobacter vinelandii mannuronan C-5-epimerase gene (algG) is part of an alg gene cluster physically organized in a manner similar to that in Pseudomonas aeruginosa; Rehm BH et al.; Alginate is an unbranched polysaccharide composed of the two sugar residues beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G) . The M/G ratio and sequence distribution in alginates vary and are of both biological and commercial significance . We have previously shown that a family of highly related mannuronan C-5-epimerase genes (algE1 to -E5) controls these parameters in Azotobacter vinelandii, by catalyzing the Ca2+-dependent conversion of M to G at the polymer level . In this report, we describe the cloning and expression of a new A . vinelandii epimerase gene (here designated algG), localized 29 nucleotides downstream of the previously described gene algJ . Sequence alignments show that algG does not belong to the same class of genes as algE1 to -E5 but that it shares 66% sequence identity with a previously described mannuronan C-5-epimerase gene (also designated algG) from Pseudomonas aeruginosa . A . vinelandii algG was expressed in Escherichia coli, and the enzyme was found to catalyze epimerization in the absence of Ca2+, although the presence of the cation stimulated the activity moderately . Surprisingly, all activity was blocked by Zn2+ . P . aeruginosa AlgG has been reported to contain an N-terminal export signal sequence which is cleaved off during expression in E . coli . This does not happen with A . vinelandii AlgG, which appears to be produced at least partly in an insoluble form when expressed at high levels in E . coli . DNA sequencing analyses of the regions flanking algG suggest that the gene is localized in a cluster of genes putatively involved in alginate biosynthesis, and the organization of this cluster appears to be the same as previously described for P . aeruginosa.

FEBS Lett, 1996 Sep 30, 394(2), 179 - 82
Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity; Yoshihara E et al.; We report here our discovery that protein D2 of the outer membrane of Pseudomonas aeruginosa is a novel porin bearing protease activity . Homogeneously purified protein D2 hydrolyzed several synthetic peptides according to the Michaelis-Menten kinetics . A specific serine protease inhibitor, diisopropyl fluorophosphate (DFP), inactivated the protease activity and {3H}DFP covalently labeled protein D2 . We tested the effect of two monoclonal antibodies raised against protein D2 on the protease activity . One antibody lowered the protease activity to about 20%, while the other enhanced it to about 300% of that without antibody . In addition, the fractions derived from the outer membrane of the protein D2-deficient mutants showed negligible protease activity, whereas similarly fractionated outer membrane proteins of the protein D2-positive parent strain showed strong protease activity.

FEMS Microbiol Lett, 1996 Sep 15, 143(1), 25 - 8
Role of gyrA mutation and loss of OprF in the multiple antibiotic resistance phenotype of Pseudomonas aeruginosa G49; Pumbwe L et al.; A clinical isolate of Pseudomonas aeruginosa G48, became resistant during fluoroquinolone treatment giving rise to the post-therapy isolate, G49 . To determine whether mutation in gyrA gave rise to fluoroquinolone resistance, G49 was transformed with a plasmid encoding gyrA (pNJR3-2); this reduced the MIC of fluoroquinolones for G49 two-fold . DNA sequencing of gyrA of G49 demonstrated a mutation at Thr-83, substituting with isoleucine . The outer membrane of G49 was shown to lack OprF, suggesting that loss of this protein may be involved in the multiple antibiotic resistance phenotype; however, when G49 was transformed with a plasmid encoding oprF (pRW5), expression of oprF was shown to have no effect upon the phenotype.

Biochem Biophys Res Commun, 1996 Sep 13, 226(2), 555 - 60
The Pseudomonas aeruginosa fumc and soda genes belong to an iron-responsive operon; Polack B et al.; Pseudomonas aeruginosa contains two superoxide dismutases (SOD), a Mn-containing SOD (Mn-SOD) and a Fe-SOD, which are encoded by sodA and sodB, respectively . We have cloned and sequenced a DNA fragment from P . aeruginosa, strain CHA, which contains the sodA gene and three other open reading frames (ORF) . We report here that one of the ORFs upstream from sodA is fumC, which encodes the O2.- resistant isoform of fumarase (or fumarate hydratase) . It is shown that fumC and sodA belong to the same operon . By primer extension experiments, the transcription initiation site has been located at -413 from the ATG codon of the fumC gene . The fumC-sodA operon was found to be negatively regulated in presence of iron and the E . coli FUR protein was shown to bind to the 19-bp FUR consensus sequence present at the transcription start site of the operon.

Lancet, 1996 Sep 7, 348(9028), 639 - 42
Spread of beta-lactam-resistant Pseudomonas aeruginosa in a cystic fibrosis clinic; Cheng K et al.; BACKGROUND: Pseudomonas aeruginosa colonisation of the airways of patients with cystic fibrosis (CF) is associated with considerable respiratory morbidity . Although segregation of colonised patients from non-colonised patients to prevent cross-infection has been recommended, there is little evidence that such cross-infection is widespread . We observed that a high proportion of children attending our CF clinic were colonised with P aeruginosa that was resistant to ceftazidime and other beta-lactam antibiotics . We used two genomic fingerprinting techniques to see whether this may have arisen from epidemic spread of a single strain . METHODS: The prevalence of P aeruginosa colonisation and the antibiotic susceptibility of the organisms was determined from review of laboratory reports in the case-notes of 120 children with CF . Isolates were cultured from the sputum of 65 children colonised with ceftazidime-resistant P aeruginosa . Polymorphisms in total bacterial DNA from 92 isolates were analysed with two molecular fingerprinting techniques--pulsed-field gel electrophoresis after restriction enzyme digestion and assessment of flagellin gene polymorphisms by amplification of the whole gene and restriction enzyme digestion . RESULTS: 92 (76.7%) of 120 children were colonised with P aeruginosa, and 65 of the 92 harboured isolates that were resistant to ceftazidime . Only three of the 92 children had never been treated with ceftazidime . The results of the two molecular-fingerprinting techniques were concordant and showed that 55 of 65 children harboured the same epidemic strain . This strain was resistant to ceftazidime, azlocillin, and imipenem, and sensitive to tobramycin and ciprofloxacin . INTERPRETATION: This study provides the first molecular evidence of a long-term outbreak of P aeruginosa in a CF centre . We suggest that careful surveillance of the prevalence of antibiotic resistance in CF centres should be instituted with measures to prevent cross-infection . We believe that antipseudomonal monotherapy should be considered with caution.

Nature, 1996 Sep 5, 383(6595), 86 - 9
The C5a chemoattractant receptor mediates mucosal defence to infection; Hopken UE et al.; A family of G-protein-coupled chemoattractant receptors is known to mediate the transport and activation of neutrophils and macrophages This family includes receptors for chemokines, such as interleukin-8, bacterial formylated peptides, platelet-activating factor, leukotriene B4, and the complement anaphylatoxins . The apparent redundancy of these receptors suggests that they have an important underlying role in host defence . To isolate the contribution of particular molecules, we disrupted a gene that encodes a single chemoattractant receptor . Here we show that mice deficient in the chemoattractant C5a receptor, in comparison to their wild-type littermates, were unable to clear intrapulmonary-instilled Pseudomonas aeruginosa, despite a marked increase in neutrophil influx, and succumbed to pneumonia . These C5a-receptor-deficient mice challenged with sublethal inocula of Pseudomonas become superinfected with secondary bacterial strains . We conclude that the C5a receptor has a non-redundant function, and is required for mucosal host defence in the lung.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9839 - 43
The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa; Whitchurch CB et al.; Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate . AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified . Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae . Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility . However, FimS does not appear to be required for alginate production in mucoid strains . FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems . The alternative sigma factor AlgU also affects both alginate production and twitching motility . Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9414 - 9
Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase; Mouz N et al.; The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines . To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P . aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker . The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain . It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase . The dodecameric organization of catabolic P . aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).

Minerva Anestesiol, 1996 Sep, 62(9), 281 - 7
{Effects of hyperbaric oxygenation in skin and pulmonary infections caused by Pseudomonas aeruginosa}; Marmo M et al.; About 80% of nosocomial infections are caused by aerobic bacteria . The Pseudomonas aeruginosa is a Gram-negative bacterium pertaining to the Pseudomonadaceae family . P . aeruginosa is responsible for 6-22% of all hospital infections . The aim of this paper is to evaluate the efficacy of both hyperbaric oxygen-therapy (HBO 2 Atm x 35 min/day) alone for 8 days and when associated to the chemoantibiotic therapy (amikacine 15 mg/kg/day for 8 days intraperitoneal), in rats infected through pulmonary and subcutaneous intake . In rats affected by P . aeruginosa, HBO induces a significant reduction in mortality and morbility with bacteria eradication in blood culture findings, bronchial aspirate and skin biopsies . These effects were increased by the use of amikacine which is an antibiotic used for the treatment of Gram-negative bacteria.

Epidemiol Mikrobiol Imunol, 1996 Sep, 45(3), 119 - 22
{The effect of pharmacodynamic parameters of pefloxacin on elastase and protease activity in Pseudomonas aeruginosa}; Hostacka A; The effect on Pseudomonas aeruginosa virulence factors (elastase and proteinase) after short-time treatment (30 min) with suprainhibitory (2x or 4x MIC) as well as with supra-subinhibitory concentrations (2x MIC + sub-MICs or 4x MIC + sub-MICs) of pefloxacin was studied . An effective decrease of elastase activity (to 42.6% of the control value) was observed only after higher suprainhibitory antibiotic concentration . More significant depression of the activities tested was found after the postantibiotic effect of subinhibitory concentrations . Elastase as well as proteinase activity were suppressed by supra-subinhibitory concentration of 4x MIC + 0.1x MIC to 13.1% or 37.9%, as compared to controls.

PDA J Pharm Sci Technol, 1996 Sep-Oct, 50(5), 306 - 10
Removal of lipid A and Pseudomonas aeruginosa endotoxin from dialysis fluids by high-flux polysulfone ultrafilter (dialyzer); Rafiee-Tehrani M et al.; Ultrafilters (dialysis membranes) are generally considered to be impermeable to bacterial endotoxins (lipopolysaccharides) contaminating dialysates used for hemodialysis therapy, due to the self-aggregating properties of its molecules in aqueous media . The aim of the present investigation was to monitor the efficiency of high-flux polysulfone ultrafilters in removing lipid A and lipopolysaccharide (LPS) from dialysis fluids . To evaluate the safety of high-flux polysulfone membranes, an in-vitro circulation system (measuring lipopolysaccharide and lipid A penetrate from the ultrafilter to the other side and vice-versa), was utilized . Peritoneal dialysis solution was spiked with various concentration of Pseudomonas aeruginosa lipopolysaccharide (LPS) as well as lipid A diphosphoryl (E . coli F-583) . Endotoxin and lipid A concentrations were detected by a chromogenic limulus amoebocyte lysate (LAL) assay . This investigation indicates that polysulfone ultrafiltration represents an efficient system to obtain an endotoxin and lipid A free dialysate when contaminated peritoneal solution was circulated through the dialysis membrane.

Plasmid, 1996 Sep, 36(2), 95 - 111
Conservation of the genetic switch between replication and transfer genes of IncP plasmids but divergence of the replication functions which are major host-range determinants; Thorsted PB et al.; The trfA operon of broad-host-range IncP plasmids is essential to activate the origin of vegetative replication in diverse species . The trb operon encodes most of the apparatus for mating pair formation, the first step in conjugative transfer . Comparison of the nucleotide sequence of the IncP beta plasmid R751 presented here with the equivalent IncP alpha sequence identifies conserved features of the organization and regulation of the trfA operon and the region controlling expression of the trb operon . As in IncP alpha plasmids, these operons are transcribed from a bidirectional promoter region consisting of trfAp for the trfA operon and trbAp and trbBp for the trb operon . The KorA-dependent switch between the trfA and trbA promoters is conserved as is the trbA gene encoding the third IncP global regulator . The intergenic region between trbA and trbB shows very little sequence identity between the two plasmids but the spacing, the KorB operator, the trbB promoter, and the existence of a hairpin loop (albeit of different actual sequence) which sequesters the trbB ribosome binding site are all conserved . The trfA operon encodes two ORFs . The first ORF is highly conserved and encodes a putative single-stranded DNA binding protein (Ssb) . The second, trfA, contains two translational starts as in the IncP alpha plasmids, generating related polypeptides of 406 (TrfA1) and 282 (TrfA2) amino acids . TrfA2 is very similar to the IncP alpha product, whereas the N-terminal region of TrfA1 shows very little similarity to the equivalent region of IncP alpha TrfA1 . This region has been implicated in the ability of IncP alpha plasmids to replicate efficiently in Pseudomonas aeruginosa . A TcR derivative of R751 was constructed and shown not to establish itself efficiently in P . aeruginosa at 37 degrees C, although it did establish itself inefficiently at lower temperatures, underlining the importance of this region in the adaptation of the plasmid to the host.

Changgeng Yi Xue Za Zhi, 1996 Sep, 19(3), 253 - 7
Squamous cell carcinoma of the ampulla of Vater: a case report; Chen CM et al.; A wide variety of neoplastic lesions may involve the ampulla of Vater, but squamous cell carcinoma has not been reported . Here we report a case of squamous cell carcinoma of papilla Vater . A 72-year-old Chinese female was manifested by obstructive jaundice and biliary tract infection . Duodenoscopic study revealed a polypoid mass with deep and broad ulcer on the ampulla of Vater . Histological examination revealed a metastatic squamous cell carcinoma . The gynecologist had examined the patient carefully and revealed a negative tumor survey . Other sources were also intensively studied but which revealed nothing . The patient developed biliary tree infection after endoscopic retrograde cholangiopancreatic study so that percutaneous transhepatic cholangiography & drainage was performed . Pseudomonas aeruginosa and Enterococcus were cultured from the drainaged bile . Radiotherapy was used and the bleeding was halted temporarily but the patient later expired due to persistent bleeding.

J Pharm Pharmacol, 1996 Sep, 48(9), 981 - 4
Mechanism for synergism between sulphonamides and trimethoprim clarified; Richards RM et al.; Pseudomonas aeruginosa, Escherichia coli, Pseudomonas cepacia and Moraxella catarrhalis were selected for their markedly different resistance patterns to sulphonamides and trimethoprim . In addition, strains of E . coli and P . cepacia were selected having different resistance profiles to the inhibition of dihydropteroate synthetase and dihydrofolate reductase . All inhibitors of dihydropteroate synthetase combined in any combination with inhibitors of dihydrofolate reductase resulted in mutual enhancement of bacterial uptakes of the inhibitors and corresponding increased antibacterial activity of the combinations . High concentrations of sulphonamides or p-aminobenzoic acid plus trimethoprim caused a decrease in overall activity of the combination and indicated that both sulphonamides and p-aminobenzoic acid at high concentrations can interact with dihydrofolate reductase . The antibacterial activity of p-aminobenzoic acid at high concentrations is considered to be a blocking effect on dihydrofolate reductase even though p-aminobenzoic acid at low concentrations is an essential part of the synthesis of dihydrofolic acid . These findings support an alternative hypothesis for the mechanism of antibacterial action of individual antifolates and their mechanism of synergism in combination.

Mol Microbiol, 1996 Sep, 21(6), 1137 - 46
A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS; Latifi A et al.; In Pseudomonas aeruginosa, the production of many virulence factors and secondary metabolites is regulated in concert with cell density through quorum sensing . Two quorum-sensing regulons have been identified in which the LuxR homologues LasR and RhlR are activated by N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL) respectively . The lasR and rhlR genes are linked to the luxl homologues lasl and rhll, which are responsible for synthesis of OdDHL and BHL, respectively . As lasRl and rhlRl are both involved in regulating synthesis of exoenzymes such as elastase, we sought to determine the nature of their interrelationship . By using lacZ transcriptional fusions in both homologous (P . aeruginosa) and heterologous (Escherichia coli) genetic backgrounds we provide evidence that (i) lasR is expressed constitutively throughout the growth cycle, (ii) rhlR expression is regulated by LasR/OdDHL, and (iii) that RhlR/BHL regulates rhll . We also show that expression of the stationary-phase sigma factor gene rpoS is abolished in a P . aeruginosa lasR mutant and in the pleiotropic BHL-negative mutant PANO67 . Furthermore, our data reveal that kin E . coli, an rpoS-lacZ fusion is regulated directly by RhlR/BHL . Taken together, these results indicate that P . aeruginosa employs a multilayered hierarchical quorum-sensing cascade involving RhlR/BHL and LasR/OdDHL, interlinked via RpoS, to integrate the regulation of virulence determinants and secondary metabolites with adaptation and survival in the stationary phase.

Ann Trop Paediatr, 1996 Sep, 16(3), 203 - 6
Community-acquired Pseudomonas aeruginosa infection in an infant; Hendricks MK et al.; A 7-month-old infant presented at a tertiary centre with a 6-day history of a skin rash, fever and diarrhoea . Clinical features included pyrexia, kwashiorkor, extensive ulcerating skin lesions suggestive of ecthyma gangrenosum, hepatomegaly, meningismus, neutropenia and iron deficiency anaemia . Blood and skin aspirate cultures yielded a positive growth of Pseudomonas aeruginosa . Apart from severe protein energy malnutrition, no other causes of immunodeficiency were found . He responded well to parenteral antibiotic therapy with gentamicin and piperacillin.

Alcohol Clin Exp Res, 1996 Sep, 20(6), 967 - 72
In vitro alcohol dehydrogenase-mediated acetaldehyde production by aerobic bacteria representing the normal colonic flora in man; Jokelainen K et al.; Excessive ethanol consumption has been related with the development of liver cirrhosis, as well as with rapid intestinal transit time and diarrhea . Moreover, heavy drinking is associated with an increased incidence of cancer of the oropharynx, larynx, esophagus, and colorectum . Acetaldehyde of microbial origin has recently been suggested as a possible pathogenic factor behind this alcohol-associated gastrointestinal morbidity . The present in vitro study was aimed to investigate alcohol dehydrogenase activity and acetaldehyde formation capacity of some major aerobic bacteria representing the normal colonic flora in man . Cytosolic alcohol dehydrogenase activity and cytosolic protein concentration were determined spectrophotometrically . Alcohol dehydrogenase activity was then calculated as nmoles of reduced substrate produced by milligrams of protein per minute . The ability of different bacteria to produce acetaldehyde was determined by incubating the intact bacterial suspension in closed vials containing ethanol (final concentration 22 mM) for 1 hr at 37 degrees C . The acetaldehyde formed during the incubation was analyzed by headspace gas chromatography . Marked differences in the alcohol dehydrogenase activity and acetaldehyde forming capacity were found among the strains tested . The alcohol dehydrogenase activity varied from 606 +/- 91 nmol/min/mg protein (Escherichia coli IH 50546) to 1 +/- 0.2 nmol/min/mg protein (E . coli IH 50817), and acetaldehyde formation varied from 1,717 +/- 2 nmol acetaldehyde/10(9) colony-forming units (Klebsiella oxytoca IH 35403) to 5 +/- 2 nmol acetaldehyde/10(9) colony-forming units (Pseudomonas aeruginosa ATCC 27853) . There was a statistically significant correlation (r = 0.77; p < 0.001) between alcohol dehydrogenase activity and acetaldehyde production from ethanol, strongly suggesting the catalytic role of bacterial alcohol dehydrogenase in this reaction.

Mol Microbiol, 1996 Sep, 21(5), 1019 - 28
Exotoxin A production in Pseudomonas aeruginosa requires the iron-regulated pvdS gene encoding an alternative sigma factor; Ochsner UA et al.; Exotoxin A (ETA) is secreted by Pseudomonas aeruginosa under iron-limiting growth conditions . The ETA structural gene, toxA, is regulated at the transcriptional level by the gene products of the regAB operon . The expression of both toxA and regAB is repressed under iron-replete conditions, suggesting a role for the ferric uptake regulator (Fur) in regulation of ETA synthesis; however, the Fur protein does not interact directly with the toxA or the regAB promoters . Evidence is presented that the iron control of ETA synthesis is mediated by a Fur-regulated alternative sigma factor, PvdS, which had initially been identified as a positive activator for the production of the siderophore pyoverdin . In a delta pvdS deletion mutant, ETA was produced at low levels of less than 5% compared to wild type, but still in response to iron starvation, and introduction of a functional pvdS gene on a plasmid fully restored wild-type levels and normal iron regulation of ETA synthesis . Therefore, a functional pvdS locus is essential for ETA production . Neither toxA nor regAB mRNA was detectable in a delta pvdS mutant . Overexpression of pvdS from the tac promoter on a plasmid resulted in a high-level and iron-independent production of ETA in wild-type PAO1, in the delta pvdS strain, but not in a delta regA strain as a host . These findings suggest that PvdS is required for the activation of the regAB promoters . The transcription of regAB and toxA after induction of the P tac-pvdS gene was monitored in cells grown in high-iron medium . While both regAB and toxA were highly expressed during all growth phases under microaerobic conditions, toxA transcripts were detected only during the exponential but not the early stationary phase of growth under aerobic conditions . These results suggest that a second regulatory mechanism besides the Fur-PvdS system is involved in iron regulation of ETA production.

Mol Microbiol, 1996 Sep, 21(5), 1001 - 17
Ferric uptake regulator mutants of Pseudomonas aeruginosa with distinct alterations in the iron-dependent repression of exotoxin A and siderophores in aerobic and microaerobic environments; Barton HA et al.; Because the ferric uptake regulator (fur) appears to be an essential gene in Pseudomonas aeruginosa, resistance to manganese was used as an enrichment to isolate strains carrying point mutations in the fur gene in order to assess its role in the co-ordinate expression of siderophores and exotoxin A (ETA) . This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, which carry point mutations in the fur gene . Two parental strains were used in this study . Three mutants were isolated from the widely used strain, PAO1 . One of these, CS (cold sensitive), has a mutation in the 5' non-coding region of the fur gene while the two other mutants derived from this parent have mutations resulting in the following deduced changes in Fur: mutant A2, H86-->R; mutant A4, H86-->Y . The other mutant (C6) and its revertant (C6Rv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of arginine deiminase and nitrate reduction) that controls anaerobic respiration in P . aeruginosa . Fur from the C6 mutant has an A10-->G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has been changed to Ser at this position . All mutants were examined for alterations in the iron-regulated expression of siderophores and ETA . The A2 and A4 mutants expressed higher levels of siderophores in iron-deficient media and in iron-replete media . The CS mutant constitutively expressed siderophores at 25 degrees C . At 42 degrees C siderophore biosynthesis was iron repressed as in the parental strain PAO1 . The deletion of anr in PAO6261 had no apparent effect on the iron-mediated regulation of siderophore synthesis, but the C6 mutant derived from this strain produces siderophores constitutively . The iron-regulated production of siderophores by C6Rv was similar to the parental strain PAO6261 and PAO1 . Because one of the parental strains used in this study is an Anr mutant, regulation of ETA production was assessed under aerobic and microaerobic conditions . Iron-dependent repression of ETA synthesis in both parental strains and A2 and A4 mutants was found to be 50-100-fold under aerobic and microaerobic conditions, as assayed by quantitative Western dot-blot assays . By contrast in the CS and C6 mutants, while iron-dependent repression os ETA synthesis was similar to both parental strains under aerobic conditions, ETA production in these mutants was constitutive in a microaerobic environment . RNase protection analysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mutant corroborated the results of quantitative dot-blot assays of ETA . The mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS) that positively regulates the expression of siderophores and ETA . Fur from the A2 and A4 mutants and from the C6Rv revertant was able to bind to the target DNA, but with reduced affinity by comparison to wild-type Fur . Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter region of the pvdS gene, but it retained some weak binding activity in gel mobility shift assays . The data presented in this study not only furnish some additional insights into the structure-function relationships of Fur, but also afford novel perspectives of virulence factors in P . aeruginosa under environmental conditions that have not previously been considered.

Clin Infect Dis, 1996 Sep, 23(3), 532 - 7
Antibiotic susceptibility of multiply resistant Pseudomonas aeruginosa isolated from patients with cystic fibrosis, including candidates for transplantation; Saiman L et al.; Chronic lung disease caused by antibiotic-resistant Pseudomonas aeruginosa in patients with cystic fibrosis (CF) is difficult to treat, especially in those who are lung transplantation candidates . Analysis of antibiotic susceptibility and synergy studies of 1,296 isolates revealed that 172 (13.3%) were multiply resistant (i.e., resistant to two or more classes of anti-Pseudomonas agents) . beta-Lactam agents (including imipenem and aztreonam) or aminoglycosides inhibited only 11% of the multiply resistant strains, while ciprofloxacin inhibited 34% . High concentrations of tobramycin and gentamicin (200 micrograms/mL), achievable by aerosol administration, inhibited 95% of isolates and overwhelmed permeability-resistance mechanisms . Antimicrobial pairs tested in checkerboard dilutions of clinically achievable drug concentrations inhibited 75% of the multiply resistant strains . On average, three additive and 2.4 synergistic pairs of antimicrobial agents had activity per strain . Transplantation candidates were older than nontransplantation candidates (P = .034), and isolates from transplantation candidates were less likely to be inhibited by antibiotic combinations (P < .001) . Administration of aerosolized aminoglycosides and synergy testing of antimicrobial combinations may represent viable therapeutic options for patients with CF.

Antimicrob Agents Chemother, 1996 Sep, 40(9), 2021 - 8
Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression; Poole K et al.; The region upstream of the multiple antibiotic resistance efflux operon mexA-mexB-oprM in Pseudomonas aeruginosa was sequenced, and a gene, mexR, was identified . The predicted MexR product contains 147 amino acids with a molecular mass of 16,964 Da, which is consistent with the observed size of the overexpressed mexR gene product . MexR was homologous to MarR, the repressor of MarA-dependent multidrug resistance in Escherichia coli, and other repressors of the MarR family . A mexR knockout mutant showed a twofold increase in expression of both plasmid-borne and chromosomal mexA-reporter gene fusions compared with the MexR+ parent strain, indicating that the mexR gene product negatively regulates expression of the mexA-mexB-oprM operon . Furthermore, the cloned mexR gene product reduced expression of a plasmid-borne mexA-lacZ fusion in E . coli, indicating that MexR represses mexA-mexB-oprM expression directly . Consistent with the increased expression of the efflux operon in the mexR mutant, the mutant showed an increase (relative to its MexR+ parent) in resistance to several antimicrobial agents . Expression of a mexR-lacZ fusion increased threefold in a mexR knockout mutant, indicating that mexR is negatively autoregulated . OCR1, a nalB multidrug-resistant mutant which overproduces OprM, exhibited a greater than sevenfold increase in expression of a chromosomal mexA-phoA fusion compared with its parent . Introduction of a mexR knockout mutation in strain OCR1 eliminated this increase in efflux gene expression and, as expected, increased the susceptibility of the strain to a variety of antibiotics . The nucleotide sequences of the mexR genes of OCR1 and its parental strain revealed a single base substitution in the former which would cause a predicted substitution of Trp for Arg at position 69 of its mexR product . These data suggest that MexR possesses both repressor and activator function in vivo, the activator form being favored in nalB multidrug-resistant strains.

Cornea, 1996 Sep, 15(5), 533 - 6
Povidone-iodine (betadine) in the treatment of experimental Pseudomonas aeruginosa keratitis; Michalova K et al.; Topical 5% povidone-iodine for the treatment of corneal ulcers was observed in Sierra Leone, West Africa by one of us (D.J.D.) . To test the efficacy of topical 5% povidone-iodine for infectious keratitis, experimental Pseudomonas aeruginosa keratitis was induced in 12 rabbits by first abrading the central 3 mm of corneal epithelium . Thirty milliliters of broth of P . aeruginosa strain ATCC 27835 (1.8 x 10(7) viable bacteria) was dropped twice on the wounded cornea . After 22 h, all corneas were clinically infected . Eight rabbits were treated with 5% povidone-iodine solution and four with 0.9% NaCl solution . All were given hourly drops . Twenty-four hours after treatment began, the central 8-mm button of the infected cornea was excised, homogenized, and serial dilutions plated onto MacConkey agar . The total number of viable Pseudomonas organisms was calculated . The treatment group had 5.2 +/- 0.4 CFUs (colony-forming units) per cornea . The control group had 4.8 +/- 0.4 CFUs per cornea (p = 0.11) . The clinical scores (Hobden grading system) were 6.9 +/- 1.5 for the treated group and 7.3 +/- 2.5 for the control group (p = 0.74) . There was no statistical difference between the treated and control groups . Povidone-iodine (5%) is not effective in the acute treatment of P . aeruginosa keratitis in this rabbit model.

Ophthalmology, 1996 Sep, 103(9), 1483 - 92
Orbital infections in patients with human immunodeficiency virus infection; Kronish JW et al.; BACKGROUND: Opportunistic infections frequently involve the anterior and posterior segments of the eye but rarely occur in the orbit in patients with human immunodeficiency virus (HIV) infection . The authors managed eight patients with HIV and unilateral orbital infections who presented between July 1988 and March 1995 . METHODS: Records of the patients were reviewed . A literature review of orbital infections in patients infected with HIV also was conducted . RESULTS: There were five men and three women, with a mean age of 33.8 years . The mean CD-4 cell count from five of the eight patients was 18.4 cells/mm3 . Invasive aspergillosis was the most common orbital infection occurring in four patients, all of whom had contiguous sinus involvement and intracranial extension . Orbital cellulitis with subperiosteal abscesses secondary to ethmoiditis caused by Propionibacterium acnes and Pseudomonas aeruginosa developed in two patients . Orbital cellulitis and panophthalmitis secondary to Staphylococcus aureus endogenous endophthalmitis developed in one patient, and one patient had presumed syphilitic optic neuritis, orbital periostitis, and necrotizing vasculitis . Five patients had permanent visual loss, including four who had loss of light perception . Four patients died of orbital diseases within 1 year of presentation, and three deaths were attributed to intracranial spread of Aspergillus fumigatus . Other organisms reported in the literature that caused orbital infections in patients with HIV include Rhizopus arrhizus, Toxoplasma gondii, and Pneumocystis carinii . CONCLUSION: Opportunistic infections of the orbit from bacterial, fungal, and parasitic organisms should be recognized as a serious complication of systemic HIV infection and are associated with a high ocular morbidity and mortality rate.

Bull Math Biol, 1996 Sep, 58(5), 923 - 38
A pharmacodynamic model for the action of the antibiotic imipenem on Pseudomonas aeruginosa populations in vitro; Berg PH et al.; The standard method for measuring in vitro antibiotic efficacy is based on a point observation of bacterial activity 18 hours after inoculation . The method, while simple, forgoes significant information by ignoring the dynamics of the interactions between antibiotic and bacteria . This paper proposes a simple dynamic model describing these interactions . The model consists of two non-linear differential equations of the S-system type . Its parameter values are estimated, through the minimization of residual errors, from data on the effect of the carbapenem antibiotic imipenem on Pseudomonas aeruginosa . The model adequately describes the dynamic behavior of the bacterial populations in the presence of the antibiotic: beginning with drug administration, then through the decline of the bacterial population and possibly ending with bacterial resurgence.

Microbiology, 1996 Sep, 142 ( Pt 9), 2333 - 40
Environmental gasoline-utilizing isolates and clinical isolates of Pseudomonas aeruginosa are taxonomically indistinguishable by chemotaxonomic and molecular techniques; Foght JM et al.; A total of 42 Pseudomonas aeruginosa strains was isolated previously from clinical sources (27 strains) and from a gasoline-contaminated aquifer (15 strains) . Selected strains were subjected to taxonomic tests involving chemical and molecular biological techniques, including membrane fatty acid analysis, phage-sensitivity, growth temperature range, presence of plasmids, and PCR-amplification and sequencing of a species-specific 16S-23S rDNA internal transcribed spacer region . The clinical and environmental isolates formed a coherent taxonomic group with few distinguishing characteristics . Of the phenotypes observed, a consistent difference was the ability of the aquifer strains to utilize gasoline supplied in the gas phase as sole carbon source and, conversely, the inability of the clinical strains to do so . Fourteen of the 15 environmental strains possessed similar-sized cryptic plasmids . The clinical isolates either lacked detectable plasmids or contained plasmids of a different size . The observation that the clinical and environmental isolates of P . aeruginosa were taxonomically indistinguishable is discussed in terms of its relevance to environmental-regulatory guidelines because P . aeruginosa, a known opportunistic pathogen, is a prime candidate for use in bioremediation processes involving deliberate release of this organism to the environment.

Invest Ophthalmol Vis Sci, 1996 Sep, 37(10), 2081 - 8
Immunoglobulin A antibodies against Pseudomonas aeruginosa in the tear fluid of contact lens wearers; Cheng KH et al.; PURPOSE: Pseudomonas aeruginosa is the most important cause of contact lens-associated ulcerative keratitis, especially for those who use extended-wear lenses . Until now, the presence of specific anti-P . aeruginosa immunoglobulin A (IgA) antibodies in the tears of contact lens wearers has not been investigated and is the purpose of the current study . METHODS: The levels of specific IgA antibodies against P . aeruginosa and total secretory IgA (s-IgA) concentrations were measured in tears of various groups of contact lens and non-contact lens wearers using enzyme-linked immunosorbent assays . Contact lens groups were divided into the following categories: daily-wear rigid gas-permeable lenses (n = 23), daily-wear soft lenses (n = 22), extended-wear soft lenses (n = 17), and non-contact lens wearers (n = 23) . As a positive control group, we tested tears obtained from patients with cystic fibrosis (n = 5) because the respiratory tract of these persons often are colonized by P . aeruginosa . RESULTS: The percentage of nonresponders (< 15 U/ml) varied between 9% in daily-wear rigid gas-permeable contact lens users to 23% in daily-wear soft contact lens users . The percentage of nonresponders in controls was 13% . The frequency of nonresponders was not significantly different among the different groups tested . All patients with cystic fibrosis showed a very high anti-P . aeruginosa IgA response in their tears . When analyzing the mean anti-P . aeruginosa IgA response, a significantly lower level was found in extended-wear contact lens users (38 U/ml) compared to non-contact lens wearers (82 U/ml) . Total s-IgA levels in the tears of the various groups tested were not significantly different . CONCLUSIONS: A substantial number of persons in the population of contact lens wearers tested lack detectable IgA antibodies against P . aeruginosa in their tears and may be susceptible to P . aeruginosa keratitis if the physiological condition of their cornea is compromised.

FEMS Microbiol Lett, 1996 Sep 1, 142(2-3), 301 - 7
Purification and characterization of LasR as a DNA-binding protein; You Z et al.; In Pseudomonas aeruginosa, the activator protein LasR and a cognate autoinducer (AI) are required for expression of the elastase gene (lasB) . In the present study, we investigated the binding properties of the P . aeruginosa lasR gene product . The LasR protein was overexpressed and purified as a glutathione S-transferase (GST) fusion protein . Using gel retardation and UV cross-linking analysis, we demonstrated that the GST-LasR could bind to a separate site in the lasB upstream operator regions 1 and 3 in the presence of the autoinducer . Regions 1 and 3 are located at 105 and 42 base pairs upstream, respectively, from the lasB transcriptional start site . Our present results clearly demonstrate that LasR is a specific DNA-binding protein that regulates the transcription of the lasB gene in the presence of an autoinducer.

J Bacteriol, 1996 Sep, 178(18), 5550 - 4
The uvrB gene of Pseudomonas aeruginosa is not DNA damage inducible; Rivera E et al.; The uvrB gene of Pseudomonas aeruginosa has been isolated from a genomic library by complementation of an Escherichia coli uvrB mutant . The complete nucleotide sequence of P . aeruginosa uvrB consists of 2,013 bp, encoding a polypeptide of 670 amino acids . A P . aeruginosa SOS consensus region, which functions as a binding site for the LexA repressor molecule, is not present in the upstream region of the uvrB gene isolated . By transcriptional fusions with a reporter gene, it has been demonstrated that, contrary to what happens with the homologous gene of E . coli, the P . aeruginosa uvrB gene is not DNA damage inducible . Nevertheless, the UvrB protein must be functional in P . aeruginosa cells because a uvrB-defective mutant is extremely sensitive to UV radiation.

J Bacteriol, 1996 Sep, 178(18), 5361 - 9
PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa; Dean CR et al.; PfeR (Regulator) and PfeS (Sensor), members of the superfamily of so-called two-component regulatory protein pairs, are required for the enterobactin-inducible production of the ferric enterobactin receptor (PfeA) in Pseudomonas aeruginosa . A pfeR knockout mutant failed to demonstrate enterobactin-inducible expression of a pfeA-lacZ fusion, indicating that PfeR acts at the level of pfeA gene expression . Consistent with this, PfeR overexpressed in P . aeruginosa bound, in bandshift assays, the promoter region of pfeA . Such binding was enhanced when PfeR-containing extracts were prepared from cells cultured in the presence of enterobactin, consistent with a model of PfeR as an enterobactin-responsive activator of pfeA expression . A region showing homology to the consensus binding sequence for the global iron repressor Fur was identified upstream of pfeR, suggesting that the pfeRS operon is iron regulated . As expected, expression of a pfeR-lacZ fusion in P . aeruginosa was increased under conditions of iron limitation . Enterobactin failed, however, to provide any enhancement of pfeR-lacZ expression under iron-limiting conditions, indicating that PfeR does not positively regulate pfeRS expression . A pfeA knockout mutant demonstrated enterobactin-inducible expression of a pfeA-lacZ fusion, indicating that the receptor is not required for the enterobactin inducibility of pfeA gene expression . Such mutants show growth, albeit reduced, in enterobactin-supplemented iron-limiting minimal medium, indicating that a second route of uptake across the outer membrane exists for ferric enterobactin in P . aeruginosa and may be important for the initial induction of pfeA in response to enterobactin.

Arch Biochem Biophys, 1996 Sep 1, 333(1), 267 - 74
Superoxide production by the mycobacterial and pseudomonad quinoid pigments phthiocol and pyocyanine in human lung cells; Gardner PR; The quinoid pigments pthiocol, produced by Mycobacterium tuberculosis, and pyocyanine, produced by Pseudomonas aeruginosa, were examined for their effects on O2.- production in cultured human lung epithelial-like A549 cells . Intracellular O2.- levels were measured using the O2.-sensitive aconitase(s), and rates of O2.- generation were assessed from rates of antimycin-resistant respiration . Elevated O2.- was detected in cells exposed to < 25 microM phthiocol and < 2 microM pyocyanine in neutral pH medium, and both agents impaired cell growth . The O2.- scavenging manganoporphyrin, Mn(III)TMPyP, partially protected cells against pyocyanine and phthiocol-mediated growth inhibition . O2.- production by phthiocol and pyocyanine was enhanced by acidification of the growth medium . Surprisingly, the dicumarol-inhibitable quinoid detoxification enzyme DT-diaphorase was a significant source of phthiocol and pyocyanine-mediated O2.- generation in cells . O2.- production in macrophages by the phthiocol analog, menadione, was shown to impair macrophage mitochondrial respiration and bactericidal activity toward Escherichia coli . Phthiocol and pyocyanine, by producing O2.-/H2O2, and inhibiting host cell aconitase activity, energetics, and other host cell functions, may contribute to the pathogenicity of M . tuberculosis and P . aeruginosa.

Am J Kidney Dis, 1996 Sep, 28(3), 415 - 9
Peritonitis associated with exit site and tunnel infections; Gupta B et al.; We reviewed all episodes of peritonitis associated with exit site and/or tunnel infection (n = 87; rate, 0.1/yr; 13% of all peritonitis episodes) occurring from 1979 to 1995 . The exit site or tunnel infection was diagnosed at the time or shortly after the patient presented with peritonitis in 66% of the episodes . In the other one third the exit site or tunnel infection was diagnosed a median of 40 days prior to the development of peritonitis . Staphylococcus aureus accounted for 52% of episodes . Pseudomonas aeruginosa was the next most common organism . In 63 (72%) of the episodes the catheter was removed to resolve the infection at a median of 8 days (range, 0 to 226 days) from the onset of peritonitis . Catheter removal after 5 days predominately for refractory peritonitis (n = 23; median time to removal, 8 days) or relapsing peritonitis (n = 11; median time to catheter removal, 103 days) . Patients with relapsing peritonitis suffered two to four episodes prior to removal of the catheter . Patients with peritonitis associated with tunnel infection were more likely to lose their catheter than patients with peritonitis associated with exit site infection (86% v 58%), while Staphylococcus epidermidis infections were less likely to result in catheter loss compared with all other organisms (15% v 82%) . After a protocol to reduce S aureus catheter infections was implemented in 1990, the rate of catheter-related peritonitis decreased from 0.14/yr to 0.05/yr due to a decrease in S aureus episodes . We conclude that peritonitis episodes associated with a tunnel infection infrequently resolve without catheter removal . Delayed catheter removal in such circumstances often results in refractory or relapsing peritonitis . Therefore, catheter removal should be done promptly . Antibiotic prophylaxis for S aureus can reduce catheter-related peritonitis.

Appl Environ Microbiol, 1996 Sep, 62(9), 3391 - 8
Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production; Winteler HV et al.; The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa . Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts . We explored the usefulness of this system for the construction of expression vectors . The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter . Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA . On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P . aeruginosa . The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR . In P . aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter . The P . aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid . Semianaerobic static cultures of P . aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid . Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen . In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P . aeruginosa during oxygen-limited growth and stationary phase.

J Infect Dis, 1996 Sep, 174(3), 537 - 43
Immunoprophylaxis against klebsiella and pseudomonas aeruginosa infections . The Federal Hyperimmune Immunoglobulin Trial Study Group; Donta ST et al.; To determine if passive immunization could decrease the incidence or severity of Klebsiella and Pseudomonas aeruginosa infections, patients admitted to intensive care units of 16 Department of Veterans Affairs and Department of Defense hospitals were randomized to receive either 100 mg/kg intravenous hyperimmune globulin (IVIG), derived from donors immunized with a 24-valent Klebsiella capsular polysaccharide plus an 8-valent P . aeruginosa O-polysaccharide-toxin A conjugate vaccine, or an albumin placebo . The overall incidence and severity of vaccine-specific Klebsiella plus Pseudomonas infections were not significantly different between the groups receiving albumin and IVIG . There was some evidence that IVIG may decrease the incidence (2.7% albumin vs . 1.2% IVIG) and severity (1.0% vs . 0.3%) of vaccine-specific Klebsiella infections, but these reductions were not statistically significant . The trial was stopped because it was statistically unlikely that IVIG would be protective against Pseudomonas infections at the dosage being used . Patients receiving IVIG had more adverse reactions (14.4% vs . 9.2%).

J Bacteriol, 1996 Sep, 178(17), 5215 - 21
Regulation of glycerol metabolism in Pseudomonas aeruginosa: characterization of the glpR repressor gene; Schweizer HP et al.; The operons of the glp regulon encoding the glycerol metabolic enzymes of Pseudomonas aeruginosa were hitherto believed to be positively regulated by the product of the glpR regulatory gene . During nucleotide sequence analysis of the region located upstream of the previously characterized glpD gene, encoding sn-glycerol-3-phosphate dehydrogenase, an open reading frame (glpR) was identified which encodes a protein of 251 amino acids that is 59% identical to the Glp repressor from Escherichia coli and could be expressed as a 28-kDa protein in a T7 expression system . Inactivation of chromosomal glpR by gene replacement resulted in constitutive expression of glycerol transport activity and glpD activity . These activities were strongly repressed after introduction of a multicopy plasmid containing the glpR gene; the same plasmid also efficiently repressed expression of a glpT-lacZ+ transcriptional fusion in an E . coli glpR mutant . Analysis of the glpD and glpF upstream region identified conserved palindromic sequences which were 70% identical to the E . coli glp operator consensus sequence . The results suggest that the operons of the glp regulon in P . aeruginosa are negatively regulated by the action of a glp repressor.

J Biol Chem, 1996 Aug 30, 271(35), 21239 - 42
The role of specific lysine residues in the passage of anions through the Pseudomonas aeruginosa porin OprP; Sukhan A et al.; When grown under phosphate-limiting conditions Pseudomonas aeruginosa expresses the phosphate-specific porin OprP . In order to determine whether any of the lysine residues located in the amino-terminal half of the protein play a role in the transport of anions through the channels, the first nine amino-terminal lysine residues of OprP were substituted with glutamates . The mutant proteins were purified and the channels they formed were characterized by reconstituting the purified porins in planar lipid membranes . In comparison to the wild-type protein, the Lys74, Lys121, and Lys126 mutants all displayed reduced levels of conductance at KCl concentrations below 1 M, and the Lys74 and Lys121 mutants no longer exhibited a saturation of conductance at high anion concentrations . In addition, the ability of phosphate ions to inhibit the conductance of Cl- ions through the channels formed by the Lys121 mutant was greatly reduced, while their ability to inhibit the Cl- conductance of the Lys74 mutant was reduced by approximately 2-fold . To clarify the roles that Lys74, Lys121, and Lys126 play in regulating the channel characteristics of OprP, these amino acids were replaced with either glycine or glutamine residues . Analysis of these mutants suggested that both Lys74 and Lys126 may serve to funnel anions toward the binding site, but only the presence of Lys121 is required for the formation of the inorganic phosphate-specific binding site of OprP.

J Immunol, 1996 Aug 15, 157(4), 1613 - 9
Escherichia coli and Pseudomonas aeruginosa induce expansion of V delta 2 cells in adult peripheral blood, but of V delta 1 cells in cord blood; Kersten CM et al.; Human peripheral blood T cells proliferate in response to Escherichia coli and Pseudomonas aeruginosa . We observed that during the first few days after stimulation a large percentage of the responding PBMC were gamma delta T cells . In our study we characterized the early T cell responses of freshly isolated adult and newborn PBMC to soluble preparations of heat-killed E . coli and P . aeruginosa . Specimens from all healthy adults tested showed intense proliferation in response to both bacterial preparations; at 6 days, the responding cells were mainly T cell blasts, of which high percentages (up to 80%) were gamma delta T cells, most expressing V delta 2/V gamma 9 . All newborn blood specimens tested also showed T cell proliferative responses, which included a marked expansion of gamma delta T cells, mainly of the V delta 1 subset . Populations of purified V delta 1 and V delta 2 T cells were obtained from adult PBMC following stimulation with E . coli; both subsets proliferated upon rechallenge with the bacterial preparations . Protease treatment of the bacterial preparations did not appreciably affect their ability to induce expansion of gamma delta T cells in either adult or cord blood, indicating that the stimulatory components were not proteins . The response of gamma delta T cells from newborns indicates that prior exposure to bacterial products is not necessary and suggests that gamma delta T cells are important elements in natural immunity to these extracellular organisms.

Biochim Biophys Acta, 1996 Aug 13, 1290(3), 231 - 40
Binding of myeloperoxidase to bacteria: effect on hydroxyl radical formation and susceptibility to oxidant-mediated killing; Britigan BE et al.; Neutrophils form superoxide anion (O2.-) and hydrogen peroxide (H2O2) and release myeloperoxidase (MPO) during ingestion of microbial pathogens . MPO, which adheres to some bacteria, catalyzes the formation of HOCl from H2O2, thereby enhancing H2O2/O2.- microbicidal activity . Hydroxyl radical (HO.), also is an important contributor to H2O2 and O2.- microbicidal activity . MPO decreases iron-catalyzed HO . production but also leads to HO . production through the reaction of O2.- and HOCl . We hypothesized that binding of MPO to bacteria could alter the magnitude and site of HO . production upon organism exposure to O2.-/H2O2 . Incubation of MPO with Escherichia coli and Pseudomonas aeruginosa resulted in stable association of MPO with the bacteria which enhanced their susceptibility to killing by O2.-/H2O2 . In the absence of MPO preincubation exposure of E . coli, but not P . aeruginosa to O2.-/H2O2, led to iron-catalyzed HO . generation . This was associated with different amounts of redox active iron in the two types of bacteria . MPO preincubation slightly decreased HO . detected with E . coli, but markedly increased HO . formation with P . aeruginosa . This likely resulted from decreased iron-catalyzed HO . production counterbalanced by increased iron-independent HO . formation . MPO preincubation did not effect bacterial killing by a system which generated only H2O2, precluding MPO-dependent HO . formation . These data are consistent with a possible role for MPO-derived HO . in the augmentation of bacterial killing by this enzyme.

J Med Chem, 1996 Aug 2, 39(16), 3107 - 13
De novo antimicrobial peptides with low mammalian cell toxicity; Javadpour MM et al.; De novo antimicrobial peptides with the sequences: (KLAKKLA)n, (KLAKLAK)n (where n = 1,2,3), (KALKALK)3, (KLGKKLG)n, and (KAAKKAA)n (where n = 2,3), were prepared as the C-terminus amides . These peptides were designed to be perfectly amphipathic in helical conformations . Peptide antibacterial activity was tested against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus . Peptide cytotoxicity was tested against human erythrocytes and 3T3 mouse fibroblasts . The 3T3 cell testing was a much more sensitive test of cytotoxicity . The peptides were much less lytic toward human erythrocytes than 3T3 cells . Peptide secondary structure in aqueous solution, sodium dodecylsulfate micelles, and phospholipid vesicles was estimated using circular dichroism spectroscopy . The leucine/alanine-containing 21-mers were bacteriostatic at 3-8 microM and cytotoxic to 3T3 cells at about 10 microM concentrations . The leucine/alanine- or leucine/glycine-containing 14-mers and the leucine/glycine 21-mer were bacteriostatic at 6-22 microM but had much lower cytotoxicity toward 3T3 cells and higher selectivities than the natural antimicrobial peptides magainin 2 amide and cecropin B amide . The 7-mer peptides are devoid of biological activity and of secondary structure in membrane mimetic environments . The 14-mer peptides and the glycine-containing 21-mer show modest levels of helicity in model membranes . The leucine/alanine-containing 21-mer peptides have substantial helicity in model membranes . The propensity to alpha-helical conformation of the peptides in amphipathic media is proportional to their 3T3 cell cytotoxicity.

Genetika, 1996 Aug, 32(8), 1068 - 73
{Interaction between Pseudomonas aeruginosa phages-transposons: genetic analysis of the trait of inhibition by prophage D3112 of phage B39 development}; Gerasimov VA et al.; A novel, previously unknown cip gene of Pseudomonas aeruginosa phage D3112 was discovered . The cip gene is responsible for inhibiting the intracellular growth of the related heteroimmune phage B39 of P . aeruginosa . It was shown that the cip gene product inhibits replication of the B39 genome, interacting, apparently, with the definite s+ site in the phage B39 genome . Mutants cip- of phage D3112, incapable of inhibiting the growth of phage B39, and B39 mutants, insensitive to the action of the cip gene product of phage D3112, were isolated.

Vaccine, 1996 Aug, 14(12), 1111 - 7
Safety and immunogenicity of a Pseudomonas aeruginosa outer membrane protein I vaccine in human volunteers; von Specht BU et al.; The outer membrane protein I (OprI) of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni2+ chelate-affinity chromatography . After safety and pyrogenicity evaluation in animals, four groups of seven adult human volunteers were vaccinated three times at four week intervals with either 500 micrograms, 200 micrograms, 50 micrograms or 20 micrograms of OprI adsorbed onto Al(OH)3 . All vaccinations were well tolerated and no systemic side effects were detected . A significant rise of antibody titers against OprI could be measured in the serum of all volunteers who had received the 500 micrograms, 200 micrograms or 50 micrograms doses . Elevated antibody titers against OprI could still be measured 30 weeks after the final vaccination . Binding of the complement component C1q to the elicited antibodies could be demonstrated, showing the ability of the latter to promote antibody-mediated complement-dependent opsonization.

Mol Microbiol, 1996 Aug, 21(4), 713 - 24
Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa; Poole K et al.; OprJ, overproduced in nfxB multidrug-resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug-resistant strain K385, were demonstrated to be immunologically cross-reactive using an OprJ-specific monoclonal antibody . Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N-terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity . The N-terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp Kpnl fragment and a 10 kbp BamHl fragment . Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three-gene operon, mexC-mexD-oprJ . The predicted mexC-mexD-oprJ gene products exhibit homology to the MexA-MexB-OprM components of the multidrug-resistance efflux pump of P . aeruginosa (43-46% identity) . Consistent with an implied role for mexC-mexD-oprJ in drug efflux, the mexC-mexD-oprJ-hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug 'exclusion' was abrogated by energy inhibitors . The mexC and oprJ products are putative lipoproteins of a molecular mass of 40,707 and 51,742 Da, respectively, while mexD was predicted to encode a protein of 111 936 Da . Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes . Overproduction of OprJ and the attendant multiple-antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87-->R change in the predicted NfxB polypeptide . OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC-mexD-oprJ expression . Consistent with this, the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli . nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression . These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC-mexD-oprJ, encoding components of a second efflux pump in P . aeruginosa.

J Antimicrob Chemother, 1996 Aug, 38(2), 287 - 91
In-vitro interactions of DX-8739, a new carbapenem, meropenem and imipenem with amikacin against multiresistant Pseudomonas aeruginosa; Giamarellos-Bourboulis EJ et al.; In order to investigate the antimicrobial interactions against multiresistant Pseudomonas aeruginosa, thirty-seven strains resistant to antimicrobial agents of five different chemical classes were exposed in vitro to the combination of three carbapenems . DX-8739, a novel DHP-I stable analogue, meropenem and imipenem with amikacin . The tested combinations expressed an enhanced killing activity against 38-46% of strains and an additive effect against 5-13% . These effects were the same whether the applied carbapenem was DX-8739, meropenem or imipenem; they were also independent of the MIC of any antimicrobial.

J Chemother, 1996 Aug, 8(4), 261 - 5
Mobilization of antibiotic resistance for transfer in Pseudomonas aeruginosa; Lesicka-Hupkova M et al.; We describe a phenomenon of mobilization of antibiotic resistance from non-transferring strains of Pseudomonas aeruginosa by cultivation with strains of P.aeruginosa capable of transferring determinants of antibiotic resistance to a susceptible recipient strain by triparental cross . In this report we described two strains of P.aeruginosa capable to mobilize for transfer the resistance determinants in strains of P.aeruginosa with multiply antibiotic resistance which were not themselves transferable to Pseudomonas recipient strains . Two strains of P.aeruginosa No . 282 and 283 from Bata's Hospital in Zlin, Czech Republic, were used as donor strains for experiments in triparental crosses . They were resistant to carbenicillin (CAR), kanamycin (KAN), cefalotin-cefazolin (CFR), cefotaxime (CTX), ceftazidime (CAZ) and aztreonam (AZA) . Both strains transferred CFR resistance to Escherichia coli K-12 rif+ recipient, but not to P.aeruginosa PAO recipients . In a second system, a strain of P.aeruginosa No . 76 from Frankfurt University Clinics was used as a donor strain . It transfers CAR resistance to PAO-1670 rif+, but not to E . coli K-12 rif+ . Two strains of P.aeruginosa from Frankfurt University Clinics No . 76 and 229 were used as intermediary recipient strains . They were resistant to CAR, KAN, CTX, CAZ, AZA, imipenem (IMP) and ofloxacin (OFL) . Strain No . 229 did not transfer any antibiotic resistance to any of both final recipient strains . Strain No . 76 transfers, as indicated, CAR resistance determinant to PAO 1670 rif+ recipient strain . Strains of E . coli K-12 No . 3110 rif+ and P.aeruginosa PAO 1670 rif+ were used as final recipient strains.

J Small Anim Pract, 1996 Aug, 37(8), 364 - 6
Prevalence of bacteria in the conjunctival sac and on the eyelid margin of clinically normal cats; Espinola MB et al.; Samples from the palpebral conjunctive, third eyelid and eyelid margin from 50 asymptomatic cats were analysed . Sixty-seven per cent of the samples showed bacterial growth with a high predominance of the genus Staphylococcus (97.8 per cent) . The most frequent species was S epidermidis (45.7 per cent), followed by S simulans (23.9 per cent), S auricularis (17.4 per cent) and S saprophyticus (6.5 per cent) . Three samples of S aureus (6.5 per cent) and one of Pseudomonas aeruginosa (2.1 per cent) were also identified . The role of these microorganisms in the eyes of cats is discussed and the importance of the new species Staphylococcus felis is stressed.

J Laryngol Otol, 1996 Aug, 110(8), 770 - 5
Malignant otitis externa in HIV and AIDS; Hern JD et al.; Malignant otitis externa is a necrotising infection of the external ear canal which may spread to include the mastoid and petrous parts of the temporal bone, leading to skull base osteomyelitis . It is almost exclusively caused by infection with Pseudomonas aeruginosa, and usually occurs in elderly non-insulin-dependent diabetic patients . However isolated cases have been reported in a small number of non-diabetic patients, particularly in children who are immunocompromised due to malignancy, malnutrition and severe anaemia . In 1984 a case of malignant otitis externa was reported in a child with an acquired immunodeficiency syndrome (AIDS)-like illness, prior to identification of the human immunodeficiency virus (HIV) . Since that time further sporadic cases of this invasive infection have been reported in HIV and AIDS . We present two further cases and also a review of the current literature.

Eur Respir J, 1996 Aug, 9(8), 1601 - 4
Lung function in bronchiectasis: the influence of Pseudomonas aeruginosa; Evans SA et al.; Sputum isolation of Pseudomonas aeruginosa (PA) is associated with extensive disease in bronchiectasis . It is not known, however, whether infection with P . aeruginosa is the result or the cause of severe disease . We compared spirometry in patients with bronchiectasis before and after infection with P . aeruginosa, with that of patients infected by other organisms . All patients (n=12) with chronic colonization by P . aeruginosa (PA group) were studied . These were compared with other patients with bronchiectasis with no isolations of P . aeruginosa (n=37, non-PA group) . In the PA group, forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) were lower than in the non-PA group . The PA group, however, also had lower values at the time of initial colonization with P . aeruginosa than the current values for the non-PA group . Change in FEV1 and FVC over time was faster in the PA group than in the non-PA group . Reduction of FEV1 and FVC over time in the PA group prior to P . aeruginosa colonization was intermediate, not being statistically different from either value above . Our results confirm the association of chronic P . aeruginosa colonization with poor lung function, but conclude that patients with bronchiectasis who become colonized by P . aeruginosa have poorer lung function when first colonized than those colonized by other organisms . Decline in lung function is faster in those chronically colonized by P . aeruginosa than in those colonized by other organisms . It is not clear whether chronic P . aeruginosa colonization causes an accelerated decline in lung function or whether it is simply a marker of those whose lung function is already declining rapidly.

Mol Microbiol, 1996 Aug, 21(3), 459 - 70
Protein secretion by heterologous bacterial ABC-transporters: the C-terminus secretion signal of the secreted protein confers high recognition specificity; Duong F et al.; Pseudomonas aeruginosa releases several extracellular proteins which are secreted via two independent secretion pathways . Alkaline protease (AprA) Is released by its own specific secretion machinery which is an ABC-transporter . Despite sequence similarities between components of ABC-transporters in different bacteria, each transporter is dedicated to the secretion of a particular protein or a family of closely related proteins . Heterologous complementation between ABC-transporters for unrelated polypeptides can occur, but only at a very low level . We show that the 50 C-terminal amino acids of AprA constitute an autonomous secretion signal . By heterologous complementation experiments between the unrelated alpha-haemolysin (HlyA) and Apr secretion systems we demonstrated that it is only the recognition of the secretion signal by the translocator which confers specificity to the secretion process . Secretion was size-dependent . However inclusion of glycine-rich repeats from HlyA in AprA seems to overcome the size limitation exerted by the Apr secretion apparatus such that the machinery secreted a hybrid protein 20 kDa larger than the normal maximal size.

J Paediatr Child Health, 1996 Aug, 32(4), 323 - 6
The prognosis of cystic fibrosis in the Western Cape region of South Africa; Westwood AT; OBJECTIVE: To study the prognosis of cystic fibrosis (CF) in South Africa . METHODOLOGY: Retrospective chart review of 102 children with CF over 20 years . RESULTS: Survival at 18 years was 54% (95% confidence intervals 30-79) . Prognosis was not influenced by gender or genotype but survival was noted to be worse for Cape Coloured children than for European children . This appeared mainly to be due to the death of eight Cape Coloured Infants, four of whom presented with oedema and anaemia, mimicking kwashiorkor . Infection with Pseudomonas aeruginosa occurred earlier in Cape Coloured children than European children (median, 1 vs 4 years) and they had a worse 5-year survival (58 vs 89%, P < 0.05) after infection . Of the 40 children born in the second decade studied, six Coloured and no European children died . CONCLUSIONS: Ethnic differences in prognosis exist for children with CF in South Africa and are probably related to underrecognition of CF and the socio-economic status of some patients.

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