J Antimicrob Chemother, 1990 Dec, 26 Suppl F, 157 - 63 New techniques for assessing pathogenicity of quinolone-resistant bacteria; Gemmell CG et al.; Ciprofloxacin and ofloxacin-resistant variants of Staphylococcus aureus and Pseudomonas aeruginosa have been compared with their original drug-sensitive progenitor strains with respect to their susceptibility to opsonophagocytosis by human polymorphonuclear leucocytes . For strains exhibiting low level resistance to each quinolone some depression of chemiluminescence (respiratory burst) was recognized whereas for strains exhibiting high level resistance some potentiation of chemiluminescence occurred . There was no difference in terms of the phagocytic ingestion of each of the strains . This study provides only limited evidence to suggest that changes in bacterial virulence are associated with the development of resistance to the quinolone antimicrobials. Pathol Biol (Paris), 1990 Dec, 38(10), 968 - 74 {Mechanism of action of proteinases of Pseudomonas aeruginosa}; Saulnier J et al.; Of the two proteases produced by Pseudomonas aeruginosa, one whose optimal pH is neutral, exhibits elastolytic activity . This elastase is produced as a prepropeptide, subsequently modified by proteolysis, and finally secreted as an active enzyme . Both proteases act mainly on hydrophobic aminoacids . The most important site for the elastase seems to be the P'1 site where Phe, Tyr and Leu residues enhance hydrolysis . The alkaline protease is less specific of this site . At least four aminoacids are needed to obtain measurable rates of hydrolysis . A synthetic substrate, Ala-Ala-Phe-Ala, is proposed for conductimetric measurements of Pseudomonas aeruginosa elastase activities in the nanomolar range . A review of recent studies shows that a very wide range of proteins can be hydrolyzed by the two Pseudomonas aeruginosa proteases, a fact which may explain why these enzymes are major determinants of the bacteria's infectivity. J Appl Bacteriol, 1990 Dec, 69(6), 871 - 6 Heterotrophic plate counts and the isolation of bacteria from mineral waters on selective and enrichment media; Manaia CM et al.; The heterotrophic plate counts of 15 brands of bottled non-carbonated mineral waters were examined and found to be generally high and variable . Four selective or enrichment media for the enumeration of coliforms (m-Endo LES and m-lauryl sulphate agar) and Pseudomonas aeruginosa (cetrimide-nalidixic acid agar and malachite green broth) were used to isolate several species of Gram-negative bacteria . Strains identified as CDC gr IVc-2 and Comamonas (Ps.) acidovorans were the two most commonly isolated . Considerable variation in populations was seen between the brands, as well as between two batches of the same mineral water. Otolaryngol Head Neck Surg, 1990 Dec, 103(6), 918 - 25 Permeability of the round window membrane to horseradish peroxidase in experimental otitis media; Kim CS et al.; The permeability of the round window membrane (RWM) was studied by using horseradish peroxidase (HRP) in the different stages of otitis media developed in guinea pigs, and it was compared with normal RWM under transmission electron microscopic examination . The experimental model of otitis media was developed by inoculation of a Pseudomonas aeruginosa suspension . When HRP could penetrate into the inner layer of a normal RWM, the duration of HRP instillation into the round window niche was 10 minutes . The permeability of the RWM in otitis media was as follows: 3-day animals showed penetration of HRP into the middle layer . In 1-week animals, HRP had penetrated into the inner layer in all 10 ears . In 2-week animals, HRP had penetrated into the inner layer in 6 of 11 ears . In 4-week animals, however, HRP was not seen in 6 of 7 ears . HRP had penetrated through the damaged focus of the epithelial cells by inflammation . These results suggest that any derangement of the epithelial linings could increase the penetration of HRP and also ototoxic materials into the inner ear through the RWM. Arch Dis Child, 1990 Dec, 65(12), 1365 - 7 Cystic fibrosis, Pseudomonas aeruginosa, and selective decontamination; Dalzell AM et al.; We used an oral topical antibiotic preparation to try and prevent oropharyngeal carriage of Pseudomonas aeruginosa in patients with cystic fibrosis . Ten of 15 patients treated with a two week course of intravenous ceftazidime together with a 90 day course of an antibiotic containing gel continued to carry P aeruginosa in the oropharynx. EMBO J, 1990 Dec, 9(13), 4323 - 9 Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria; Filloux A et al.; The xcp genes are required for protein secretion by Pseudomonas aeruginosa . They are involved in the second step of the process, i.e . the translocation across the outer membrane, after the exoproteins have reached the periplasm in a signal peptide dependent fashion . The nucleotide sequence of a 2.5 kb DNA fragment containing xcp genes showed at least two complete open reading frames, potentially encoding proteins with molecular weights of 41 and 19 kd . Products with these apparent molecular weights were identified after expression of the DNA fragment in vitro and in vivo . Subcloning and complementation experiments showed that both proteins are required for secretion . The two products are located in the inner membrane and share highly significant homologies with the PulL and PulM proteins which are required for the specific secretion of pullulanase in Klebsiella pneumoniae . These homologies reveal the existence of a common mechanism for protein secretion in Pseudomonas aeruginosa and Klebsiella pneumoniae. J Surg Res, 1990 Dec, 49(6), 476 - 82 Efficacy of right atrial infusion of PGE1 in sepsis-induced pulmonary hypertension; Marini CP et al.; In this study we investigated the effects of right atrial infusion of PGE1 (RAIPGE1) in doses from 40 to 500 ng/kg/min on sepsis-induced pulmonary artery hypertension (SIPAH) . Thirteen pigs were randomized into a time-course group (n = 6) and a PGE1-treated group (n = 7) . Pulmonary hypertension (PAH) was induced with the infusion of Pseudomonas Aeruginosa (PsAr) at a concentration of 2 X 10(8) CFU/20 kg/min in both groups . The infusion of PsAr caused a significant and persistent rise in mean pulmonary artery pressure (MPA), pulmonary vascular resistance (PVRI), right ventricular compliance (RVC), RV dp/dt, and right ventricular stroke work index (RVSWI), 30 min after the onset of infusion (P less than 0.05 vs baseline) . Systemic hemodynamics and gas exchange were not affected throughout the 3-hr period of infusion (P = NS); however, left ventricular compliance (LVC) was depressed at a MPA greater than 35 mm Hg . The RAIPGE1 following SIPAH caused a concentration-dependent reduction above 40 ng/kg/min of MPA, PVRI, RVSWI, and RV dp/dt (P less than 0.05, 120 and 500 ng/kg/min vs PAH) . RVC returned to baseline values during the infusion of PGE1 . Systemic hemodynamics, including oxygen delivery and extraction, were unaffected by the infusion of PGE1, but LVC was improved (P less than 0.05, PGE1 500 vs PAH) . The infusion of PGE1 caused a concentration-dependent rise in shunt fraction (Qs/Qt) and alveolararterial oxygen gradients which reached statistical significance during the infusion of 500 ng/kg/min . Our data show that RAIPGE1 is effective in ameliorating RV and pulmonary hemodynamics, but at the largest dose it negatively affects gas exchange. J Bacteriol, 1990 Dec, 172(12), 7188 - 99 Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK; Totten PA et al.; Flagella in procaryotes are complex structures requiring the coordinate expression of over 50 genes, including flagellin, the major repeating structural protein . We have previously shown that a functional RpoN gene product is required for expression of flagellin in Pseudomonas aeruginosa PAK (P . A . Totten and S . Lory, J . Bacteriol . 172:389-396, 1990) and have now cloned, sequenced, and determined the transcriptional start site of the structural gene for this flagellin . The clones containing this gene produced a protein that reacted on Western immunoblots with polyclonal and four different monoclonal antibodies to purified flagella . However, this flagellin protein in Escherichia coli was slightly smaller (41 kDa) than flagellin protein produced in P . aeruginosa PAK (45 kDa), indicating degradation in E . coli or modification in P . aeruginosa . Comparison of the deduced amino acid sequence of this gene with the amino acid sequences of other flagellins revealed a conservation in the N- and C-terminal domains, suggesting conservation of secretion or assembly signals between these organisms . The sequence 5' of the structural gene contained potential RpoN-specific promoters as well as a promoter sequence recognized by RpoF (sigma 28), the alternative sigma factor required for expression of flagellin genes in E . coli (and Bacillus subtilis) . Deletion analysis of the promoter region as well as transcriptional start site mapping implicated the RpoF, and not the RpoN, consensus sequences as the functional promoter for the flagellin gene . Models for the involvement of both RpoN and RpoF in the expression of flagellin in P . aeruginosa are presented. J Bacteriol, 1990 Dec, 172(12), 6631 - 6 Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose; Bhat R et al.; Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone . The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1 . The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose . Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions . No pyrophosphate groups were detected . Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1 . Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone . All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted . Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine . The proposed lipid A structure differs from previous models . There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent. Infect Immun, 1990 Dec, 58(12), 3819 - 28 Generation and characterization of murine antiflagellum monoclonal antibodies that are protective against lethal challenge with Pseudomonas aeruginosa; Rosok MJ et al.; Two murine monoclonal antibodies, IIG5 (IgG3) and IVE8 (IgG2a), that bind to Pseudomonas aeruginosa type a flagella and type b flagella, respectively, were prepared by conventional hybridoma methodology . Specificity of each monoclonal antibody for type a or type b flagella was demonstrated by enzyme-linked immunosorbent assay, indirect immunofluorescence, and immunoblotting . The percentage of P . aeruginosa isolates recognized by each monoclonal antibody was analyzed by enzyme-linked immunosorbent assay . Among a panel of 257 flagellated P . aeruginosa clinical isolates, IIG5 bound to 67.7% of the isolates and IVE8 bound to another 30.7%, for a combined coverage of 98.4% . Inhibition of motility of P . aeruginosa by the monoclonal antibodies was observed in vitro in a soft agar assay and was dose dependent . The protective efficacy of IIG5 and IVE8 was examined in a mouse burn wound sepsis model . The antiflagellum monoclonal antibodies provided specific and significant prophylactic and therapeutic protection against lethal challenge with P . aeruginosa strains. Chem Pharm Bull (Tokyo), 1990 Dec, 38(12), 3476 - 9 Synthetic cephalosporins . VII . Synthesis and antibacterial activity of 7-{(Z)-2-(2-aminothiazol-4-yl)-2-(3-(3-hydroxy-4-pyridon-1-yl)-3- carboxypropoxyimino)acetamido}-3-(1,2,3-thiadiazol-5-yl)-thiomethyl-3- cephem-4-carboxylic acid and its related compounds; Sakagami K et al.; Synthesis and antibacterial activity of 7-{(Z)-2-(2-aminothiazol-4-yl)-2-(3-(3-hydroxy-4-pyridon-1-y l)-3- carboxypropoxyimino)acetamido}-3-(1,2,3-thiadiazol-5-yl)thio methyl-3-cephem-4-carboxylic acid (12a) and its related compounds are described . Compound 12a exhibited excellent antibacterial activity against gram-negative bacteria, including Pseudomonas aeruginosa. Yonsei Med J, 1990 Dec, 31(4), 308 - 14 Pharmacokinetics of intravitreally injected liposome-encapsulated tobramycin in normal rabbits; Kim EK et al.; Bacterial endophthalmitis, which is a devastating complication of intraocular surgery or eye trauma, has a poor prognosis . Intravitreal injection of antimicrobial agents has become a part of the standard treatment of endophthalmitis . The authors investigate the pharmacokinetics of intravitreal liposome-encapsulated tobramycin as a possible method of prolonging the duration of therapeutic concentrations . Tobramycin was encapsulated into liposomes of phosphatidylcholine, phosphatidic acid, and alpha-tocopherol by the reverse phase evaporation method . The final liposomal suspension contained tobramycin, 7.0 mg/ml, 60.5% encapsulated . One eye received an intravitreal injection of either liposome-encapsulated tobramycin (LET), tobramycin phosphated-buffered saline (TS) or a mixture of tobramycin and liposome-encapsulated saline (TEL), and the results were as follows: 1 . Concentrations of free tobramycin were significantly lower with LET than with TS or TEL at 1 hour after intravitreal injection . 2 . Concentrations of free and total tobramycin were significantly higher with LET than with TS or TEL at 5 and 8 days after intravitreal injection . Concentrations of free tobramycin with TS were lower than the minimal inhibitory concentration(MIC) of tobramycin for Pseudomonas aeruginosa at 8 days after intravitreal injection, while those with LET were higher than the MIC of tobramycin for Pseudomonas aeruginosa 18 days after injection. J Appl Physiol, 1990 Dec, 69(6), 2290 - 5 Pulmonary compliance: early assessment of evolving lung injury after onset of sepsis; Byrne K et al.; We compared the sensitivity of dynamic (Cdyn) and static lung compliance (CL) with indicators of permeability injury in a model of septic porcine adult respiratory distress syndrome . Two groups of anesthetized ventilated swine (15-25 kg) were studied . Septic animals (Ps, n = 13) received Pseudomonas aeruginosa intravenously for 1 h, which resulted in severe adult respiratory distress syndrome . Controls (C, n = 13) received 0.9% NaCl . Cdyn, CL, bronchoalveolar lavage for protein estimation, and thermal cardiogreen extravascular lung water (EVLW) measurements were performed in seven C and eight Ps animals . Six C and five Ps animals underwent gamma camera measurement of lung-to-heart ratio (slope index) of 99Tc-labeled human serum albumin . Both Cdyn and CL decreased significantly (P less than 0.01) at 30 min and thereafter in Ps vs . C . EVLW, slope index, and bronchoalveolar protein content increased significantly (P less than 0.05) in Ps vs . C at 120, 150, and 300 min, respectively . Cdyn and CL decreased well before onset of permeability injury . These early changes may be due to release of vasoactive mediators and sequestration of neutrophils in the pulmonary capillaries and later to increases in EVLW . Measurement of Cdyn and CL represents an early means of assessing evolving lung injury in this acute septic porcine model. Antimicrob Agents Chemother, 1990 Dec, 34(12), 2381 - 6 Appearance of amikacin and tobramycin resistance due to 4'-aminoglycoside nucleotidyltransferase {ANT(4')-II} in gram-negative pathogens; Jacoby GA et al.; Following the use of amikacin as the principal aminoglycoside at a Denver hospital, amikacin resistance appeared first in Pseudomonas aeruginosa and then in Escherichia coli, Klebsiella pneumoniae, and other enteric organisms from debilitated and compromised patients who had spent time in intensive care units and who had been treated with multiple antibiotics, usually including amikacin . In a P . aeruginosa isolate, resistance to amikacin and tobramycin was transferable by the IncP-2 plasmid pMG77, while in E . coli and K . pneumoniae resistance was carried by the transmissible plasmids pMG220, pMG221, and pMG222 belonging to the IncM group . Isolates and transconjugants produced an enzyme with adenyltransferase activity with substrates having a 4'-hydroxyl group, such as amikacin, kanamycin, neomycin, Sch 21768, isepamicin (Sch 21420), or tobramycin, but not with aminoglycosides lacking this target, such as dibekacin, netilmicin, sisomicin, or gentamicin C components . Genes encoding the 4'-aminoglycoside nucleotidyltransferase {ANT(4')} activity were cloned from pMG77, pMG221, and pMG222 . A DNA probe prepared from the ANT(4') found in P . aeruginosa hybridized with the ANT(4') determinant found in E . coli . A probe for the ANT(4') from Staphylococcal spp., which differs in its modification of substrates, like dibekacin, that have a 4"- but not a 4'-hydroxyl group, failed to hybridize with the gram-negative ANT(4') determinant, which consequently has been termed ANT(4')-II. J Gen Microbiol, 1990 Dec, 136 ( Pt 12), 2351 - 7 A combined physical and genetic map of Pseudomonas aeruginosa PAO; Ratnaningsih E et al.; A combined physical and genetic map of Pseudomonas aeruginosa PAO was constructed by pulsed-field gel electrophoresis and Southern hybridization using cosmid clones from a genomic library carrying known genes . A total of 37 SpeI restriction fragments have been mapped on the 5862 kb genome, and fragment contiguity demonstrated by hybridization with clones from a SpeI junction fragment library and fragments obtained by partial SpeI digestion, both derived from the P . aeruginosa PAO chromosome. J Clin Microbiol, 1990 Dec, 28(12), 2627 - 31 Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels; Fomsgaard A et al.; A silver staining method used routinely for detecting bacterial lipopolysaccharide (LPS) in sodium dodecyl sulfate-polyacrylamide gels (C . Tsai and E . Frasch, Anal . Biochem . 119:115-119, 1982) appeared to be inappropriate for visualizing certain LPS preparations . It did not stain S-form fractions of polyagglutinable Pseudomonas aeruginosa LPS or several partly deacylated (alkali-treated) S-form LPS after sodium dodecyl sulfate-polyacrylamide gel electrophoresis . However, these LPS preparations could be detected by anti-LPS sera after electroblotting onto nitrocellulose, thereby confirming their integrity and presence in the polyacrylamide gel . This is because LPS fractions containing a low number of fatty acids are washed out of the gel during the initial fixing step (40% ethanol-4% acetic acid, overnight) . By omitting this fixing step, which was originally developed for detecting proteins, and by increasing the LPS oxidation time (from 5 to 20 min), we restored the ability to detect LPS fractions that otherwise would not be stained . These modifications did not affect the detection of other S- and R-form LPSs . Thus, differences in the number of fatty acids present in polyagglutinable P . aeruginosa LPS may result in a selective loss of fatty acid-deficient S-form LPS in these apparent R-form LPS preparations . This modified procedure provides a fast, simple, and sensitive way to analyze LPS in polyacrylamide gels despite the number of acyl groups present. Eur J Biochem, 1990 Nov 26, 194(1), 109 - 18 Involvement of the hydrophobic patch of azurin in the electron-transfer reactions with cytochrome C551 and nitrite reductase; van de Kamp M et al.; The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments . In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln . Both patches were previously thought to be involved in electron transfer . 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin . Observed changes in midpoint potentials could be attributed to electrostatic effects . The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35 . Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase. Biochem J, 1990 Nov 15, 272(1), 263 - 4 Evidence for Mössbauer spectroscopy for different forms of iron core in Pseudomonas aeruginosa bacterial ferritin; Reid NM et al.; Mossbauer spectroscopic studies of whole cells of Pseudomonas aeruginosa, grown under different conditions, indicate that the predominant form of iron in the cells varies significantly . These differences are interpreted in terms of differences in the nature of the iron cores of the bacterial ferritin, which result from different growth conditions. J Biol Chem, 1990 Nov 15, 265(32), 20033 - 6 A single cyclohexadienyl dehydrogenase specifies the prephenate dehydrogenase and arogenate dehydrogenase components of the dual pathways to L-tyrosine in Pseudomonas aeruginosa; Xia TH et al.; Dual biosynthetic pathways diverge from prephenate to L-tyrosine in Pseudomonas aeruginosa, with 4-hydroxyphenylpyruvate and L-arogenate being the unique intermediates of these pathways . Prephenate dehydrogenase and arogenate dehydrogenase activities could not be separated throughout fractionation steps yielding a purification of more than 200-fold, and the ratio of activities was constant throughout purification . Thus, the enzyme is a cyclohexadienyl dehydrogenase . The native enzyme has a molecular weight of 150,000 and is a hexamer made up of identical 25,500 subunits . The enzyme is specific for NAD+ as an electron acceptor, and identical Km values of 0.25 mM were obtained for NAD+, regardless of whether activity was assayed as prephenate dehydrogenase or as arogenate dehydrogenase . Km values of 0.07 mM and 0.17 mM were calculated for prephenate and L-arogenate, respectively . Inhibition by L-tyrosine was noncompetitive with respect to NAD+, but was strictly competitive with respect to either prephenate or L-arogenate . With cyclohexadiene as variable substrate, similar Ki values for L-tyrosine of 0.06 mM (prephenate) and 0.05 mM (L-arogenate) were obtained . With NAD+ as the variable substrate, similar Ki values for L-tyrosine of 0.26 mM (prephenate) and 0.28 mM (L-arogenate), respectively, were calculated . This is the first characterization of a purified, monofunctional cyclohexadienyl dehydrogenase. Med Clin (Barc), 1990 Nov 3, 95(15), 568 - 71 {Bacteremia originated in intravascular cannulae: an epidemiologic study of 91 episodes}; Torne Cachot J et al.; This reports the analysis of an epidemiologic study of intravascular cannula bacteriemia (ICB) in a Barcelona university hospital . There were 91 episodes of ICB representing the 26.7% of the total hospital bacteriemia . In 60.6% of ICB the diagnosis was made in an intensive care area . The most common microorganisms were Staphylococcus epidermidis (27.8%), Pseudomonas aeruginosa (18.5%), and Staphylococcus aureus (14.4%) . Intravascular cannulae with higher incidence of bacteriemia were the central venous catheters (55%) and the arterial lines (29%) . Bacteriemia produced by arterial lines had short free period interval (7.7 days) and in 80% of the cases were produced by Gram negative bacteria whereas that bacteriemia produced by central venous catheters had a long free period (11.2 days) and the most frequent agents were Gram positive bacteria . The overall mortality was 17% and that attributed to the infection 6% . An age above 65 years had a mortality rate of 33% and was identified as the only significant prognostic factor (p less than 0.001) . The mean hospitalization period was 49.9 days and the cost of the treatment 830.000 ptas/patient. J Burn Care Rehabil, 1990 Nov-Dec, 11(6), 575 - 80 Infection control in a burn center; Lee JJ et al.; No consensus has been reached on the ideal isolation technique to prevent hospital-acquired infection in the patient with burns . This study reports four 2-month consecutive periods of microbial surveillance in a burn center intensive care unit . Phase I, the first period of surveillance, demonstrated a unit-acquired colonization rate of 63%, with the marker organisms appearing at 4 to 8 days . Direct observation of isolation technique showed a 51% error rate . A mandatory educational session reviewing the high colonization rates, observed breaks in isolation technique, and principles of infection control failed to decrease the colonization rates as measured in phase II . A simplified isolation technique was adopted, which led to a decrease in unit-acquired colonization, from 63% to 33% in phase III from phase I values (p = 0.0514); and to a significant delay in inception, from 7.8 to 21 days, in those colonized with Pseudomonas aeruginosa (p less than 0.05) . The simplified isolation technique decreased isolation costs over a 6-month period from $53,000 to $30,000 . To confirm the decrease colonization rates from phase I to phase III, a fourth 2-month surveillance period was undertaken 6 months later . Phase IV demonstrated similar results to those of phase III. J Antimicrob Chemother, 1990 Nov, 26 Suppl D, 137 - 42 Infection prophylaxis in neutropenic patients with acute leukaemia--a randomized, comparative study with ofloxacin, ciprofloxacin and co-trimoxazole/colistin; Arning M et al.; Preliminary results are presented of an ongoing, prospective, randomized, study comparing ofloxacin, ciprofloxacin and co-trimoxazole/colistin for the prevention of infection in patients with acute leukaemia . The results for 59 patients (median age 47 years, range 21-72) included 88 episodes of neutropenia, each associated with a course of cytotoxic therapy . The main factor measured was the time elapsed from the beginning of neutropenia (neutrophils less than 500/microliter) until the first infectious febrile episode . The median time for the period was 12 days (range 1-56) for the cotrimoxazole/colistin group, 15 days (range 1-38) for the ofloxacin group and 20 days (range 1-36) for the ciprofloxacin group (differences not significant) . Microbiologically proven major infections occurred in 10/27 treatment courses with co-trimoxazole/colistin 7/31 courses with ofloxacin and 7/30 courses with ciprofloxacin (P not significant) . These were mostly due to Gram-positive cocci . There were no Gram-negative infections in the quinolone groups compared with one major Pseudomonas aeruginosa infection in the co-trimoxazole/colistin group . No Pneumocystis carinii infections were encountered . Adverse reactions associated with co-trimoxazole/colistin required discontinuation of medication in 11/27 treatment courses because of compliance problems, skin reactions or gastrointestinal intolerance . There were significantly fewer discontinuations in the ofloxacin (n = 2) and in the ciprofloxacin groups (n = 3) . Major side effects of the quinolones included persistent icterus in one patient receiving ofloxacin and psychiatric symptoms in one patient receiving ciprofloxacin . It is concluded from these data that there were no statistically significant differences between the three treatment groups in respect of the prevention of infection.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Hosp Pharm, 1990 Nov, 47(11 Suppl 3), S3 - 6 Overview of gram-negative sepsis; Dudley MN; An overview of gram-negative sepsis is presented, and the need for improved treatment for this condition is emphasized . The availability of new and more potent antimicrobial agents has not substantially altered the mortality from sepsis and septic shock . Gram-negative infection, bacteremia, sepsis, and septic shock remain major clinical problems, particularly among hospitalized patients . The estimated incidence of gram-negative sepsis in the United States alone is 200,000 cases annually . The predominant pathogens are Escherichia coli, Klebsiella, and Pseudomonas aeruginosa . Mortality is strongly influenced by the host's clinical status and age and the development of shock; it may reach 90% in patients with rapidly fatal disease . Analysis of risk factors and use of criteria for categorizing severity of disease can be helpful in designing new treatments, identifying potential recipients of such agents, and evaluating outcome of therapy . Because bacterial endotoxin plays a pivotal role in triggering the biological cascade of mediators in the septic process, a new therapy has been developed, immunotherapy with monoclonal antibodies that neutralize lipopolysaccharide by binding to lipid A . Successful treatment of gram-negative sepsis requires appropriate patient identification and timely intervention . While antimicrobial agents remain important, monoclonal antibodies hold promise as a new therapeutic intervention. J Clin Microbiol, 1990 Nov, 28(11), 2551 - 4 Assessment of a new hub design and the semiquantitative catheter culture method using an in vivo experimental model of catheter sepsis; Segura M et al.; An in vivo model of hub-related catheter sepsis in rabbits is reported . The model was used to investigate the protection offered by a new hub design against external contamination by Pseudomonas aeruginosa or Staphylococcus epidermidis and to reassess the diagnostic value of the semiquantitative culture method in bacteremia of endoluminal origin . Contamination of conventional Luer-Lock connectors was followed by clinical sepsis, positive catheter segment cultures, or both, whereas contamination of the new hub was followed by complete protection . Clinical and bacteriological discrepancies observed between contamination with P . aeruginosa and S . epidermidis suggest that the virulence of microorganisms may account for differences in the natural history of hub-originated catheter sepsis . The semiquantitative extraluminal method for catheter culture yielded less than 15 CFU in three animals with proven bacteremia and should not be used as the "gold standard" to define catheter-related bacteremia. J Clin Microbiol, 1990 Nov, 28(11), 2520 - 5 Prospective multicenter study of vascular-catheter-related complications and risk factors for positive central-catheter cultures in intensive care unit patients; Richet H et al.; To determine the incidence rate of complications associated with vascular catheters in intensive care unit patients and to analyze risk factors for a positive vascular culture, we performed a multicenter study of intensive care unit patients at eight French hospitals . During the study period, 865 intravenous catheters were inserted in 566 patients; 362 (41.8%) were peripheral catheters, and 503 (58.2%) were central catheters . Local complications (i.e., infiltration) occurred significantly more often with peripheral than with central catheters (P less than 0.001); in contrast, fever and bacteremia were significantly more often associated with central than with peripheral catheters (P less than 0.01 and P less than 0.05, respectively) . The culture of the vascular-catheter tip was positive for 24% of central catheters (32 of 1,000 catheters days) and for 9% of peripheral catheters (21 of 1,000 catheters days) . Staphylococcus epidermidis was the most common microorganism isolated from both peripheral and central catheters, followed by Staphylococcus aureus and Pseudomonas aeruginosa . No significant risk factor associated with positive cultures for peripheral catheters was found by univariate analysis . In contrast, the purpose of the cannula (nutrition and monitoring of central venous pressure), the insertion site (jugular), the dressing type (semipermeable transparent dressing), the antiseptic used to prepare the insertion site (povidone iodine), and routine changing of the intravenous administration set were significantly associated with positive cultures of central catheters . Three factors, duration of catheterization, use of a semipermeable transparent dressing, and the jugular insertion site, were found to be independently associated with positive cultures of central catheters by multivariate analysis. Cesk Oftalmol, 1990 Nov, 46(6), 463 - 5 {Disinfection of ophthalmologic tonometers}; Beran V et al.; The authors describe an automatic instrument of their own design for disinfection of ophthalmological tonometers by germicide radiation . The tests of effectiveness against microbes (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa), spores of Bacillus mesentericus, adenoviruses and viruses of Herpes simplex provided evidence of a high disinfectant potency of the apparatus. Biochem J, 1990 Nov 1, 271(3), 565 - 9 Rapid and sensitive techniques for identification and analysis of 'reactive-centre' mutants of C1-inhibitor proteins contained in type II hereditary angio-oedema plasmas; Aulak KS et al.; Novel procedures for structural analysis of the 'reactive-centre' residues, particularly the P1 residue, of the dysfunctional C1-inhibitor proteins found in the plasmas of type II hereditary angio-oedema (HAE) patients are described . C1-inhibitor is adsorbed directly from plasma on to Sepharose-anti-(C1 inhibitor) beads . The P1 residue of C1 inhibitor is arginine and hence a potential cleavage site for trypsin . Thus trypsin digestion of the immobilized protein, followed by SDS/PAGE of the released fragments, identifies P1 residue mutations . Pseudomonas aeruginosa elastase digestion of the immobilized protein, followed by purification of the released C-terminal peptide (by h.p.l.c.) and N-terminal sequence analysis defines the new P1 residue (or other mutations in the reactive-centre region) . The techniques are both rapid and highly sensitive, requiring only 400 microliters of plasma . In addition, they permit accurate assessment of the level of normal (functional) inhibitor in a subclass of type II HAE plasmas, those containing P1-residue mutant proteins. Eur J Immunol, 1990 Nov, 20(11), 2505 - 8 J chain deficiency in human IgM monoclonal antibodies produced by Epstein-Barr virus-transformed B lymphocytes; Meng YG et al.; Six human IgM monoclonal antibodies against Pseudomonas aeruginosa were purified and characterized . On agarose-acrylamide sodium dodecyl sulfate (SDS) gels run under nonreducing conditions, IgM monoclonal antibodies showed variable amounts of a slower migrating form of IgM in addition to the one co-migrating with plasma IgM . Protein blotting with anti-J chain antibody showed that the slower migrating form did not contain J chain . Analysis of one of the monoclonal antibodies by sucrose gradient centrifugation showed that the J chain-deficient form sedimented faster than the complete IgM . It is known that IgM preparations lacking J chain sediment faster by sucrose gradient centrifugation analysis and tend to form hexamers . The slower migrating form of IgM we observed on SDS gels under nonreducing conditions could be hexameric IgM . Further evaluation of this monoclonal antibody demonstrated that both forms of IgM had the same antigen-binding activity . Glycosylation of the light chain was demonstrated in two of the monoclonal antibodies. Invest Ophthalmol Vis Sci, 1990 Nov, 31(11), 2241 - 3 Quinolones in collagen shields to treat aminoglycoside-resistant pseudomonal keratitis; Hobden JA et al.; Rabbit corneas were infected with a tobramycin-resistant (minimum inhibitory concentration, 31.25 micrograms/ml) strain of Pseudomonas aeruginosa 27853 (10(3) colony-forming units) and were treated 22 hours later with collagen corneal shields hydrated in either 25 mg/ml ciprofloxacin, 40 mg/ml norfloxacin, 40 mg/ml tobramycin, or deionized water . Shields were removed at 26 hours postinfection, and 1 hour later, corneas were harvested for bacterial enumeration . Application of shields hydrated in ciprofloxacin reduced the number of viable bacteria per cornea approximately 4 log units compared with the application of shields containing tobramycin or deionized water (P less than 0.0001) . Use of shields hydrated in norfloxacin reduced the number of P . aeruginosa organisms by greater than 2 log units compared with shields containing tobramycin or deionized water (P less than 0.0001) . Ciprofloxacin was significantly more effective than norfloxacin in reducing the number of bacteria per cornea (P less than 0.0001) . There was no significant difference in the number of bacteria recovered from corneas treated with tobramycin or deionized water (P less than 0.56). Zh Mikrobiol Epidemiol Immunobiol, 1990 Nov, (11), 19 - 21 {The assessment of the toxigenicity of clinical strains of Pseudomonas aeruginosa by an immunoenzyme method using nitrocellulose filters}; Brodinova NS et al.; The enzyme immunoassay (EIA) on nitrocellulose filters was adapted for the detection of exotoxin A in 104 P . aeruginosa strains isolated from patients and the environment in surgical wards in hospitals of Moscow and Alma-Ata . The method was shown to be highly sensitive: it permitted the detection of 5.ng of P . aeruginosa exotoxin A . The screening of 104 P . aeruginosa clinical strains by means of EIA on nitrocellulose filters revealed that these strains exotoxin A in 88.5% of cases. Cesk Epidemiol Mikrobiol Imunol, 1990 Nov, 39(6), 347 - 52 {Determination of the phospholipase C gene in Pseudomonas aeruginosa using DNA hybridization}; Burian J et al.; The authors tested the possibility to use the method of colony hybridization for screening of thermolabile haemolysin (phospholipase C) in strains of Pseudomonas aeruginosa . As a radioactively labelled probe they used SmaI fragments of the pGV26 plasmid, which contains the 6.3 kb fragment of the chromosome of strains P . aeruginosa PAD1 with the gene for phospholipase C. Zh Mikrobiol Epidemiol Immunobiol, 1990 Nov, (11), 30 - 3 {The diagnostic effectiveness of erythrocyte immunoreagents made from different Pseudomonas aeruginosa antigens}; Egizova SA et al.; The diagnostic effectiveness of the passive hemagglutination (PHA) tests with the use of erythrocyte diagnostica (ED based on exotoxin A (ETA) and poly- and monovalent ED based on lipopolysaccharides (LPS) isolated from 5 most widespread P . aeruginosa serogroups was compared . 97 patients with different purulent septic diseases and 100 practically healthy adult donors were examined . The treatment of serum samples with 2-mercaptoethanol decreased the diagnostic effectiveness of serological examination . The simultaneous determination of the activity of antibodies to ETA and LPS essentially increased the number of positive results of the PHA tests in patients with bacteriologically confirmed P . aeruginosa infection, but not in patients from whom other bacteria were isolated . Considering the sensitivity and specificity of serological examination, as well as the decrease of the volume of necessary work and the consumption of immunoreagents, the optimum test conditions proved to be ensured by the use of two ED: LPS-based polyvalent ED and ETA-based ED. Gig Sanit, 1990 Nov, (11), 32 - 3 {Use od ozone for disinfection of ships' system of water supply contaminated by Pseudomonas aeruginosa}; Rakhmanin IuA et al.; Experimental substantiation is given of the use of ozone in doses, recommended for disinfection of water and ship water supply systems infected by Pseudomonas aeruginosa . The positive effect of ozonation of water supply systems infected by Pseudomonas aeruginosa was confirmed by results of field testing on ships of the Black sea marine steam-navigation. Rev Clin Esp, 1990 Nov, 187(7), 343 - 5 {Cervical actinomycosis starting as a monoparesis of the upper left extremity}; Stiefel P et al.; We present a case of cervical actinomycosis which due to its localization, unusually low and posterior, made its debut as a monoparesis and which due to its unexpected size, marked aggressiveness, and radiologic image mimicked a tumor . We have not found in the literature this kind of presentation up to date . Although sulphur grains were not observed, empiric treatment could be started based on the Gram stain information . Culture was delayed 15 days, but we could promptly obtain reliable diagnosis thanks to pathologic study . Associated Pseudomonas aeruginosa was isolated. Mol Gen Mikrobiol Virusol, 1990 Nov, (11), 29 - 32 {Genetic determinants of tetracycline resistance of plasmids from the bacterial species Pseudomonas}; Erova TE et al.; The colony hybridization and tetracycline accumulation techniques made possible to demonstrate the majority of the 29 bacteria of Pseudomonas genera to harbour the plasmids carrying tet determinants belonging to classes A-C, while one strain contained a plasmid carrying tet determinant of class B . For the first time the determinants of classes D and E were found on R plasmids in Pseudomonas aeruginosa . The determinant of G class was identified on two plasmids identical to pBS221 plasmid described previously. J Antimicrob Chemother, 1990 Nov, 26 Suppl C, 49 - 57 Comparison of cefodizime with various cephalosporins for their indirect effect on the human neutrophil oxidative burst in vitro; Labro MT et al.; Cefodizime, a 2-amino-thiazolyl cephalosporin, is reported to display in-vitro, ex-vivo and in-vivo immunomodulatory properties; in particular, it enhances the survival of mice infected with cefodizime-resistant pathogens . We have used an in-vitro model to assess the indirect effect of this drug (compared with other cephalosporins) on the neutrophil (PMN) oxidative response . Pseudomonas aeruginosa was employed as the bacterial target for cefodizime and cefotaxime (MICs greater than 128 mg/l), cefsulodin (MIC 16 mg/l) and ceftazidime (MIC 32 mg/l) . After overnight growth in the presence of subinhibitory concentrations of each drug (10 mg/l), the altered filamentous P . aeruginosa induced a stronger oxidative response of PMN than untreated control bacteria . For all cephalosporins this was related to alterations of bacterial structure leading to increased deposits of antibodies and/or complement . Furthermore, increased non-opsonin dependent stimulation of the PMN oxidative burst was obtained; the strongest response was observed with cefodizime-treated P . aeruginosa in the case of low responder PMN, which displayed a deficient response after stimulation by control bacteria . The possibility that cefodizime could enhance this PMN function in opsonin-deficient patients requires further investigation. Antimicrob Agents Chemother, 1990 Nov, 34(11), 2142 - 7 Development of quinolone-imipenem cross resistance in Pseudomonas aeruginosa during exposure to ciprofloxacin; Radberg G et al.; Selection and regrowth of ciprofloxacin-resistant variants, which were present in low frequencies in the initial inoculum, were seen when large inocula of Pseudomonas aeruginosa were incubated with ciprofloxacin . These variants showed cross resistance to other quinolones . In 8 of 13 strains tested, ciprofloxacin selected imipenem-resistant variants in a similar way to imipenem . The opposite phenomenon of ciprofloxacin-imipenem cross resistance after exposure to imipenem was not detected . None of the ciprofloxacin-resistant variants showed cross resistance to aztreonam, piperacillin, or tobramycin . These findings indicate that widespread and uncritical use of ciprofloxacin gives a potential risk of development of resistance in P . aeruginosa not only to quinolones but also to another unrelated useful agent, imipenem . In vitro evaluation of this phenomenon in isolates from patients with P . aeruginosa infections may be justified, since strains differ in development of quinolone-imipenem cross resistance after ciprofloxacin exposure. Antimicrob Agents Chemother, 1990 Nov, 34(11), 2093 - 6 Beta-lactam-fosfomycin antagonism involving modification of penicillin-binding protein 3 in Pseudomonas aeruginosa; Reguera JA et al.; Antagonism between fosfomycin and antipseudomonal penicillins, cefotaxime, and ceftriaxone was observed in Pseudomonas aeruginosa RYC212 . Fosfomycin, a non-beta-lactam antibiotic that acts on bacterial cell wall synthesis, decreased the expression of penicillin-binding protein 3 and induced beta-lactamase . The antagonistic effect was reduced in the presence of high concentrations of the beta-lactamase inhibitor tazobactam or in fosfomycin-resistant mutants . We suggest that products resulting from fosfomycin cell wall damage could interact with a system that regulates penicillin-binding protein and beta-lactamase production. Antimicrob Agents Chemother, 1990 Nov, 34(11), 2065 - 9 Inhibition of Pseudomonas aeruginosa elastase and Pseudomonas keratitis using a thiol-based peptide; Burns FR et al.; Pseudomonas aeruginosa elastase is a zinc metalloproteinase which is released during P . aeruginosa infections . Pseudomonas keratitis, which occurs following contact lens-induced corneal trauma, can lead to rapid, liquefactive necrosis of the cornea . This destruction has been attributed to the release of both host-derived enzymes and the bacterial products P . aeruginosa elastase, alkaline protease, exotoxin A, and lipopolysaccharide endotoxin . A synthetic metalloproteinase inhibitor, HSCH2 (DL)CH{CH2CH(CH3)2}CO-Phe-Ala-NH2, which we previously showed to be a potent inhibitor of corneal collagenase and alkali-induced corneal ulceration, was tested as a potential inhibitor of P . aeruginosa elastase . Inhibition constants (Kis) for the resolved diastereomers were determined with the chromogenic substrate furylacryloyl-glycyl-L-leucyl-L-alanine . One isomer had a Ki of 0.3 microM, while the other had a Ki of 0.4 microM . The more potent diastereomer was evaluated in vivo in experimentally induced Pseudomonas keratitis in rabbits . Following inoculation of one cornea of each rabbit, topical treatment with a 1 mM solution of the inhibitor significantly delayed the onset of corneal melting and perforation, as compared with the results for the control and gentamicin-treated groups . This protective effect suggests that the inhibitor may have a therapeutic application by delaying the progression of corneal destruction in Pseudomonas keratitis. Kansenshogaku Zasshi, 1990 Nov, 64(11), 1439 - 46 {Effects of macrolide antibiotics against mucoid Pseudomonas aeruginosa}; Yokoiyama S; Macrolide antibiotics at concentrations by far lower than their MICs proved to inhibit the production of alginate, elastase, and protease by mucoid Pseudomonas aeruginosa . The morphological study of mucoid P . aeruginosa under the electron microscope revealed the slime-like structures common to the cell morphology of the organism in cultured colonies and foci in a model for respiratory tract infection in mice, and the strains of mucoid P . aeruginosa which had been allowed to get in contact with the drugs proved to produced obviously fewer slime-like structures than control strains . The effect of a 14-membered macrolide, erythromycin, against mucoid P . aeruginosa was also observed with a 16-membered macrolide, rokitamycin this effect appeared to be common to the macrolide. Can J Microbiol, 1990 Nov, 36(11), 801 - 3 Population dynamics of pseudomonads after iodination; Pyle BH et al.; The population dynamics of pseudomonads grown under rich or low nutrient conditions were examined following iodination . Iodinated and untreated controls of Pseudomonas aeruginosa or Pseudomonas cepacia were resuspended in phosphate buffer and incubated at room temperature . Viable populations of iodine-treated cultures increased faster in phosphate-buffered water than uniodinated controls . Thus, bacteria in iodinated water systems may recover or multiply during storage and distribution after disinfection and may pose a significant health risk. J Antibiot (Tokyo), 1990 Nov, 43(11), 1450 - 63 A new antipseudomonal cephalosporin CP6162 and its congeners; Iwamatsu K et al.; The synthesis and biological activity of a series of 3-{2-(5-hydroxy-4-pyridon-2-yl)ethenyl}cephalosporin derivatives are described . They showed very potent activity against Gram-negative bacteria, especially Pseudomonas aeruginosa . (6R, 7R)-7-{(Z)-2-(2-Aminothiazol-4-yl)-2 -(1-carboxy-1-methyl)-ethoxyiminoacetamido}-3-{(Z)-2-(1,5-dihydrox y-4- pyridon-2-yl)ethenyl}ceph-3-em-4-carboxylic acid, CP6162 (8e), was selected for further evaluation as antipseudomonal chemotherapeutic agent. Rev Infect Dis, 1990 Nov-Dec, 12 Suppl 8, S1055 - 64 Etiology of infection and morphologic changes in the lungs of Filipino children who die of pneumonia; Gonzaga NC et al.; Histopathologic studies and isolation of virus and bacteria in culture were carried out for 71 children less than 5 years of age with fatal pneumonia . A potential microbial etiology was identified for 61 children (86%): bacteria for 19 (27%), virus for 16 (23%), and virus plus bacteria for 26 (37%) . Staphylococcus was the most prevalent pathogen, alone or in combination with other organisms, followed by Pseudomonas aeruginosa . Viral infection may predispose to bacterial infection in some children . A correlation of clinical course, results of cultures, and morphologic changes revealed cofactors that may have contributed to a fatal outcome . Lung abscess, pericarditis, myocarditis, endocarditis, and meningitis were associated with bacterial infection . Many patients in this study had severe bronchopneumonia, with a high prevalence of complications such as abscess (62%), atelectasis (40%), pericarditis (28%), and empyema (7%) . Such complications added to multiple infections, measles, and malnutrition contributed to the fatal outcome in these children. Zentralbl Veterinarmed B, 1990 Nov, 37(9), 684 - 95 {Detection specificity of an optimal solid-phase enzyme immunoassay for Pseudomonas aeruginosa and Pseudomonas mallei}; Niederwohrmeier B et al.; The evaluation and application of an enzyme-immunoassay (EIA) for the detection of Pseudomonas (Ps.) aeruginosa and Ps . mallei is described . Polystyrene beads (1/4'') as the solid-phase are prepared by coating the balls with purified IgG from the serum of rabbits (9-12 micrograms/bead) in Coating-Buffer pH 9.6 . After washing the balls they are saturated with 10% BSA or 10% FCS in PBS-Tween 20 . The bacteria bound to the coated balls are detected by the specific peroxidase labelled IgG . This EIA using Ps . aeruginosa (P9) as a model is able to detect this bacterium within 5 hours, with stored coated balls 3.5 hours, with a detection limit of 10(4) CFU . Nine Pseudomonas-strains react stronger than other strains . These cross-reactions can be substantially reduced by absorbing the P9-conjugate with the cells of Ps . stutzeri (P15) . With the other Pseudomonas-strains a high specificity is found with the P9-conjugate . After modifying this EIA for the detection of Ps . mallei (P18) the strains Ps . mallei (P57), Ps . pseudomallei (P17) and Ps . cepacia (P67) react with the P18-conjugate . With the other tested strains a high specificity is found at 10(7) CFU . The polystyrene bead-EIA is recommended as a sensitive and specific test for the detection of Ps . aeruginosa in about 5 resp . 3.5 hours . It only requires normal laboratory equipment and is thus a highly practicable method for routine diagnostic of Ps . aeruginosa. J Infect Dis, 1990 Nov, 162(5), 1112 - 7 Comparative therapy with cefpirome alone and in combination with rifampin and/or gentamicin against a disseminated Pseudomonas aeruginosa infection in leukopenic mice; Valdes JM et al.; Treatment of disseminated Pseudomonas aeruginosa infection in leukopenic mice was evaluated using cefpirome alone and in combination with gentamicin and/or rifampin . Mice were made leukopenic with cyclophosphamide and infected through a skin incision with an inoculum of 1250 organisms (13 LD50) . Antibiotics were administered subcutaneously for 48 h . Although the addition of cefpirome to gentamicin and/or rifampin improved survival significantly at 48 h compared with untreated controls (84.6%-100% vs . 38.5%), therapy with these combinations did not improve survival significantly from that achieved with cefpirome alone . Quantitative blood and tissue (liver, spleen, kidney, lung) cultures in mice treated with cefpirome alone or including rifampin were lower than in infected controls or groups receiving therapy that excluded cefpirome . Highest counts were observed in mice receiving cefpirome plus gentamicin . Except for the cefpirome plus gentamicin group, which demonstrated areas of acute tubular necrosis, the cefpirome group had less tissue pathology than infected controls. J Bacteriol, 1990 Nov, 172(11), 6576 - 80 Role of the far-upstream sites of the algD promoter and the algR and rpoN genes in environmental modulation of mucoidy in Pseudomonas aeruginosa; Mohr CD et al.; The role of several regulatory elements in environmental modulation of mucoidy in Pseudomonas aeruginosa was studied . Transcriptional activation of algD, necessary for the mucoid phenotype, was found to depend on FUS, the newly identified far-upstream sites of the algD promoter . The FUS were delimited to a region spanning nucleotides -432 to -332 relative to the algD mRNA start site . Insertional inactivation of algR in PAO568 abolished the algD promoter response to nitrogen availability and greatly diminished but did not completely eliminate reactivity to changes in salt concentration . Insertional inactivation of rpoN (ntrA) in PAO568 did not affect algR and algD transcription. J Bacteriol, 1990 Nov, 172(11), 6396 - 402 Analysis of cloned structural and regulatory genes for carbohydrate utilization in Pseudomonas aeruginosa PAO; Temple L et al.; Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments . Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd) . Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment . The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes . In this study, hexR was mapped coincident with edd . A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes . The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer. J Bacteriol, 1990 Nov, 172(11), 6252 - 60 Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa; Mohr CD et al.; A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed . This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest . Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction . This DNA is then transferred into P . aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome . Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations . One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P . aeruginosa . The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors . This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters . Additional insertions were obtained in regions downstream and upstream of algR . A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR . This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa . Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases . An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source. J Pharm Pharmacol, 1990 Nov, 42(11), 808 - 9 Susceptibility of bacterial isolates from AIDS patients to six fluoroquinolones; Lewin CS et al.; The susceptibilities to ciprofloxacin, DR-3355 (S-(-)-ofloxacin), enoxacin, lomefloxacin, ofloxacin and PD127,391 of 69 significant bacterial isolates from HIV-positive patients at the City Hospital, Edinburgh have been determined . With the exception of the enterococci, most of the strains tested (including staphylococci, Escherichia coli and Pseudomonas aeruginosa) were susceptible to the fluoroquinolones . Ciprofloxacin was the most active of the clinically available drugs followed by ofloxacin, lomefloxacin and enoxacin . PD127,391 and DR-3355, the new fluoroquinolones tested, were at least as active as ciprofloxacin . Hence bacterial infections in AIDS patients should respond to fluoroquinolone therapy. J Hosp Infect, 1990 Nov, 16(4), 343 - 9 Pseudomonas aeruginosa orbital cellulitis in four neutropenic patients; Atkins MC et al.; Four neutropenic patients on a haematology ward developed orbital cellulitis due to different strains of Pseudomonas aeruginosa over a 7-month period . Investigation of patients and the ward environment revealed two P . aeruginosa isolates indistinguishable from the infecting strains in a plastic washing bowl and a sink in a single cubicle respectively . These items were unlikely to have been the sources of the infecting strains but were a potential cross infection hazard . Treatment of orbital cellulitis is discussed briefly. J Hosp Infect, 1990 Nov, 16(4), 311 - 8 Effect of three plastic catheters on survival and growth of Pseudomonas aeruginosa; Martinez-Martinez L et al.; The effect of polyvinylchloride (PVC), polyurethane (PU) and siliconized latex (SL) catheters on the survival and growth of six non-mucoid and three mucoid strains of Pseudomonas aeruginosa was evaluated . Pseudomonas aeruginosa (1 x 10(8)) was incubated in PBS alone (control) or with 30 1-cm length segments of each catheter and the number of viable microorganisms was determined after 8 h, 1, 2, 5, 7 and 10 days . The presence of PVC catheters significantly favoured the survival and growth of non-mucoid strains in comparison to the control (P less than 0.05 at 5 days, P less than 0.01 at 7 days and thereafter); a similar result was observed with SL catheters (P less than 0.05 at 2 days, P less than 0.01 at 5 days and thereafter) . No differences were observed with PU catheters . The number of mucoid microorganisms decreased with time in all controls and suspensions containing segments of catheter, but non-mucoid revertants appeared and quickly increased in the presence of PVC and SL (but not PU) catheters . Eluates of PBS previously containing PVC or SL segments induced a 100- to 500-fold increase in the growth of a non-mucoid strain in comparison with PBS alone . It is concluded that some plastic catheters can release substance(s) that favour the viability of P . aeruginosa. Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8889 - 93 Elimination of infectious human immunodeficiency virus from human T-cell cultures by synergistic action of CD4-Pseudomonas exotoxin and reverse transcriptase inhibitors; Ashorn P et al.; We have previously described a recombinant protein, designated CD4(178)-PE40, consisting of the human immunodeficiency virus (HIV) envelope glycoprotein-binding region of human CD4 linked to the translocation and ADP-ribosylation domains of Pseudomonas aeruginosa exotoxin A . By virtue of its affinity for gp120 (the external subunit of the HIV envelope glycoprotein), the hybrid toxin selectively binds to and kills HIV-1-infected human T cells expressing surface envelope glycoprotein and also inhibits HIV-1 spread in mixed cultures of infected and uninfected cells . We now report that CD4(178)-PE40 and reverse transcriptase inhibitors exert highly synergistic effects against HIV-1 spread in cultured human primary T cells . Furthermore, combination treatment can completely eliminate infectious HIV-1 from cultures of human T-cell lines . This conclusion is based on protection of a susceptible cell population from HIV-induced killing, complete inhibition of virus protein accumulation, and elimination of HIV DNA (as judged by quantitative polymerase chain reaction analysis) . The results highlight the therapeutic potential of treatment regimens involving combination of a virostatic drug that inhibits virus replication plus an agent that selectively kills HIV-infected cells. Clin Exp Immunol, 1990 Nov, 82(2), 289 - 93 Anti-mitochondrial antibodies (anti-M7) in heart diseases recognize epitopes on bacterial and mammalian sarcosine dehydrogenase; Klein R et al.; The anti-mitochondrial antibody (AMA) anti-M7 has been shown to occur exclusively in sera from patients with acute and chronic myocarditis . Applying different enzymes of the inner mitochondrial membrane to ELISA, anti-M7-positive sera reacted only with sarcosine dehydrogenase (SD) from Pseudomonas aeruginosa . Testing these sera in the Western blot against a commercially available SD as well as against SD prepared from rat liver mitochondria, a determinant at 42 kD and 90 kD, respectively, was visualized . Using submitochondrial particles (SMP) from bovine heart and rat liver another major determinant at 64 kD could be observed with both antigen fractions . Liver SMP also expressed the SD-related, 90-kD epitope . Sera from patients with other AMA-positive and AMA-negative autoimmune diseases were negative with these different determinants . The identity of the 64-kD epitope on heart and liver SMP as well as the 42-kD polypeptide of bacterial SD and the 90-kD epitope on mammalian SD was proven by absorption studies and by elution of antibodies from the antigen bound to the immobilon sheets after immunoblotting . The SD enzyme activity was not affected by anti-64-kD and anti-42-kD antibodies in vitro . It is concluded that anti-M7 antibodies may be stimulated by an antigen expressed on cardiocytes during an infection which shares epitopes with SD, an evolutionary highly conserved protein . SD-sensitized B cell clones could therefore be triggered by the M7-antigen which shows homology to SD. Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 455 - 61 Purification of the pyocin S2 complex from Pseudomonas aeruginosa PAO1: analysis of DNase activity; Seo Y et al.; Pyocin S2 purified from mitomycin C-induced lysates of Pseudomonas aeruginosa strain PAO1 has been shown to consist of a complex of two proteins . Further analysis of the purified S2 complex revealed that the 74 kd S2 pyocin demonstrates DNase activity which can be blocked by S2-specific antisera . Chromosomal DNA from pyocin sensitive cells treated with the pyocin S2 complex in vitro did not show any degradation, suggesting that the 10 kd protein inhibits the DNase activity of the S2 protein . These results suggest an alternative mechanism for the toxicity associated with the S2 pyocin. J Immunol Methods, 1990 Oct 19, 133(2), 263 - 8 Qualitative analysis of antibody binding . An in vitro assay for the evaluation and development of vaccines; Bruderer U et al.; We describe a rapid in vitro assay for the evaluation of in vivo properties of conjugate vaccines . Using human and murine monoclonal antibodies (mAb) specific for lipopolysaccharides (LPS), isolated from Pseudomonas aeruginosa, we determined in a competitive binding assay the amount of LPS or conjugate vaccine which was required to inhibit the antibody binding to LPS by 50% (I50 values) . Furthermore, utilizing a murine burn wound sepsis model, we determined the potential of the same conjugates to induce protection in vivo against infection with the corresponding bacteria . Protective mAb have approximately 100-fold lower I50 values for preparations which are highly effective in inducing protection than for preparations which are ineffective . Furthermore, in the case of potent preparations it was noted that protective mAb exhibit similar I50 values for the conjugates and for the corresponding LPS . These results suggest that the fast and easily interpretable in vitro assay described may significantly facilitate the development and optimization of vaccines. Biochemistry, 1990 Oct 9, 29(40), 9377 - 86 Sequential 1H NMR assignments of iron(II) cytochrome c551 from Pseudomonas aeruginosa; Detlefsen DJ et al.; Sequence-specific 1H NMR resonance assignments for all but the C-terminal Lys 82 are reported for iron(II) cytochrome c551 from Pseudomonas aeruginosa at 25 degrees C and pH = 6.8 . Spin systems were identified by using TOCSY and DQF-COSY spectra in 2H2O and 1H2O . Sequential assignments were made by using NOESY connectivities between adjacent amide, alpha, and beta protons . Resonances from several amino acids including His 16, Gly 24, Ile 48, and Met 61 experience strong ring-current shifts due to their placement near the heme . All heme protons, including the previously unassigned propionates, have been identified . Preliminary analysis of sequential and medium-range NOEs provides evidence for substantial amounts of helix in the solution structure . Long-range NOEs indicate that the folds in solution and crystal structures are similar . For one aromatic side chain (Tyr 27) that is close to the heme group we found a transition from hindered ring rotation at low temperature to rapid rotation at high temperature. Infect Immun, 1990 Oct, 58(10), 3363 - 8 Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response; Pedersen SS et al.; Alginate, a viscous polysaccharide from mucoid Pseudomonas aeruginosa, may interfere with the host defenses in patients with cystic fibrosis and chronic P . aeruginosa lung infection . The alginate concentration in the sol phase of expectorated sputum was quantitated by a biochemical method and a newly developed enzyme-linked immunosorbent assay . There was a high degree of correlation between the methods, and the concentration of alginate ranged from 4 to 101 micrograms/ml with a median of 35.5 micrograms/ml when measured by enzyme-linked immunosorbent assay . Alginate could not be detected in the bronchial secretions from patients without P . aeruginosa infection . In vitro investigation of alginate did not show any activation of the alternative pathway of complement, as determined by a hemolytic kinetic assay and by testing for neutrophil chemotaxis . At a high concentration, P . aeruginosa alginate caused a slight activation of the classical pathway of complement . Alginate did not cause neutrophil chemotaxis by itself but was able to reduce the neutrophil chemotactic response to N-formylmethionylleucylphenylalanine and for zymosan-activated serum . P . aeruginosa and seaweed alginates were able to prime neutrophils for increased N-formylmethionylleucylphenylalanine-induced neutrophil oxidative burst, as determined by chemiluminescence . Because of its ability to prevent attraction of neutrophils to the site of infection, lack of complement activation, and ability to enhance neutrophil oxidative burst, alginate from P . aeruginosa may contribute to the persistence and pathogenesis of chronic P . aeruginosa infection in cystic fibrosis. Pneumologie, 1990 Oct, 44(10), 1177 - 9 {High-grade tracheomalacia and tracheal stenosis in congenital esophageal atresia with lower esophagotracheal fistula (Type III b)}; Wiersbitzky S et al.; Stenosis and malacia of the trachea wall can provoke chronic stridor and/or chronic bronchitis, but usually stenosis and malacia only exist separately . The finding of an infant born with atresia of the oesophagus and a lower tracheoesophageal fistula which was cured by surgery on the 1st day of life are discussed . During the following 8 months we observed persistent stridor, chronic cough and (4-times) relapsing episodes of respiratory insufficiency ("nearly-sudden-infant-death-syndrome"/NSIDS) due to gastrooesophageal reflux (GER with aspiration) and severe tracheomalacia combined with tracheostenosis and bacterial infections (Pseudomonas aeruginosa) . The strategy of therapy for GER and for the tracheal abnormality are discussed. Acta Ophthalmol (Copenh), 1990 Oct, 68(5), 615 - 6 Multiple scleral abscesses with recurrent bacterial endophthalmitis eight months following cataract surgery; Ram J et al.; Scleral involvement in association with endophthalmitis is unusual . We report a case of recurrent bacterial endophthalmitis and multiple scleral abscesses occurring 8 months following cataract extraction . Culture of pus from scleral abscesses first grew Pseudomonas aeruginosa; and klebsiella pneumoniae on recurrence . The patient responded to intensive topical and systemic antibiotic therapy and recovered visual acuity of 20/60. J Antimicrob Chemother, 1990 Oct, 26 Suppl B, 83 - 9 Pefloxacin therapy for nosocomial infections in the intensive care unit; Potgieter PD; Nosocomial infections occurring in an intensive care unit (ICU) are commonly caused by aerobic Gram-negative bacilli or Staphylococcus aureus, which are frequently multi-resistant and difficult to treat and contribute significantly to the patients' morbidity in the ICU . Pefloxacin, with its wide range of antimicrobial activity, lack of serious side-effects and advantageous kinetics, is a useful drug for use in this group of critically ill patients . Pefloxacin has achieved a greater than 70% clinical cure rate and a microbiological response of over 80% in cases of nosocomial pneumonia in the ICU . Failure and superinfection has occurred with the development of resistance, particularly in Pseudomonas aeruginosa in a small number of cases, but this can be prevented by combination antimicrobial therapy . Serious side-effects, including confusion, psychiatric disturbance and other neurological abnormalities were rare and resolved on withdrawal of the drug . Drug interactions occur with cimetidine and theophylline but are usually not clinically relevant; significant interaction with warfarin occurs and the dose of warfarin needs careful adjustment . Pefloxacin is a valuable drug for use in bacteriologically proven sensitive infections and combination with aminoglycosides or beta-lactam agents should prevent the development of resistance. J Antimicrob Chemother, 1990 Oct, 26 Suppl B, 193 - 8 Pefloxacin versus ceftazidime in therapy of soft tissue infections in compromised patients; Segev S et al.; Soft tissue infections in compromised patients are frequently caused by Gram-negative organisms and particularly by Pseudomonas aeruginosa . These pathogens are effectively eradicated by pefloxacin as well as by ceftazidime . The effectiveness and safety of these two agents were compared in a prospective randomized study in 67 patients with soft tissue infections . Underlying conditions included malignant diseases, diabetes mellitus and chronic renal failure . The infections included: post operative infection, septic foot, soft tissue abscess and cellulitis . Thirty-three patients were treated with intravenous ceftazidime for a mean duration of ten days . More than half the 34 patients given pefloxacin were treated only orally for a mean period of 13 days . The clinical and bacteriological outcomes were similar in both groups . There was clinical cure or improvement in 26 pefloxacin cases and in 23 ceftazidime cases, failure in six pefloxacin cases and in seven ceftazidime and relapse in two pefloxacin and in three ceftazidime patients . The bacteriological responses were eradication in 23 pefloxacin cases and in 22 ceftazidime cases, persistence in five pefloxacin cases and in six ceftazidime cases, relapse in one pefloxacin case and in none of the ceftazidime group, reinfection in four pefloxacin cases and in three ceftazidime cases and there was one unassessed patient in the pefloxacin group and two in the ceftazidime group . Nausea and vomiting occurred in three patients and elevation of liver enzymes in another patient; all side effects were observed only in the pefloxacin treated patients . These results suggest that oral pefloxacin could offer an alternative to intravenous ceftazidime in half the compromised patients with tissue infections . However, adverse reactions due to pefloxacin administration should be watched for during such therapy. Invest Ophthalmol Vis Sci, 1990 Oct, 31(10), 1940 - 4 Ciprofloxacin iontophoresis for aminoglycoside-resistant pseudomonal keratitis; Hobden JA et al.; Studies using ciprofloxacin for the therapy of experimental aminoglycoside-resistant keratitis caused by Pseudomonas aeruginosa were conducted using transcorneal iontophoresis as the drug-delivery system . Corneas infected with P . aeruginosa ATCC 27853/pMG6 were treated 22 hours postinfection with ciprofloxacin delivered by iontophoresis (0.8 mA X 10 min), mock iontophoresis (eyecup with no current), or frequently applied topical drops . Iontophoresis of 10 mg/ml or 25 mg/ml of ciprofloxacin significantly reduced the number of viable bacteria per cornea by more than 5 log units compared with untreated controls (P less than 0.0001) . Five hours after the initiation of treatment, mock iontophoresis (10 mg/ml or 25 mg/ml) or 11 applications of topical ciproflaxicin drops (7.5 mg/ml) decreased the viable bacteria relative to the untreated controls by 5 log units (P less than 0.0001) . One treatment with an eyecup was as effective as 11 treatments with topical drops (P greater than 0.75) . One hour after treatment with iontophoresis or mock iontophoresis of 10 mg/ml of ciprofloxacin, aqueous humor concentrations were 83.75 +/- 8.85 micrograms/ml and 24.87 +/- 4.0 micrograms/ml (mean +/- standard error of the mean), respectively . One hour after the last of five applications of 7.5 mg/ml of ciprofloxacin (every 15 min for 1 hr) the aqueous humor concentration was 4.2 +/- 1.14 micrograms/ml . These results show the value of ciprofloxacin in treating aminoglycoside-resistant infections caused by P . aeruginosa and suggest that ciprofloxacin can be efficiently delivered by iontophoresis. J Antimicrob Chemother, 1990 Oct, 26 Suppl B, 207 - 13 The clinical problems of bacterial resistance to the new quinolones; Acar JF et al.; Clinical problems of bacterial resistance to the new fluoroquinolones are emerging as their use increases . Emergence of resistant strains has been observed in various types of infections, especially of the respiratory tract . Only limited studies, however, deal with strains isolated from clinical specimens . The identity between the original strain and the resistant variant is rarely proved . Resistance to quinolones can be due to a modification in DNA gyrase or to an alteration in outer membrane permeability (pleiotropic resistance) . Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa have been recognized as species at risk of developing such resistance . Strategies to minimize the emergence of resistance are discussed. J Clin Invest, 1990 Oct, 86(4), 1030 - 7 Possible role of bacterial siderophores in inflammation . Iron bound to the Pseudomonas siderophore pyochelin can function as a hydroxyl radical catalyst; Coffman TJ et al.; Tissue injury has been linked to neutrophil associated hydroxyl radical (.OH) generation, a process that requires an exogenous transition metal catalyst such as iron . In vivo most iron is bound in a noncatalytic form . To obtain iron required for growth, many bacteria secrete iron chelators (siderophores) . Since Pseudomonas aeruginosa infections are associated with considerable tissue destruction, we examined whether iron bound to the Pseudomonas siderophores pyochelin (PCH) and pyoverdin (PVD) could act as .OH catalysts . Purified PCH and PVD were iron loaded (Fe-PCH, Fe-PVD) and added to a hypoxanthine/xanthine oxidase superoxide- (.O2-) and hydrogen peroxide (H2O2)-generating system . Evidence for .OH generation was then sought using two different spin-trapping agents (5.5 dimethyl-pyrroline-1-oxide or N-t-butyl-alpha-phenylnitrone), as well as the deoxyribose oxidation assay . Regardless of methodology, .OH generation was detected in the presence of Fe-PCH but not Fe-PVD . Inhibition of the process by catalase and/or SOD suggested .OH formation with Fe-PCH occurred via the Haber-Weiss reaction . Similar results were obtained when stimulated neutrophils were used as the source of .O2- and H2O2 . Addition of Fe-PCH but not Fe-PVD to stimulated neutrophils yielded .OH as detected by the above assay systems . Since PCH and PVD bind ferric (Fe3+) but not ferrous (Fe2+) iron, .OH catalysis with Fe-PCH would likely involve .O2(-)-mediated reduction of Fe3+ to Fe2+ with subsequent release of "free" Fe2+ . This was confirmed by measuring formation of the Fe2(+)-ferrozine complex after exposure of Fe-PCH, but not Fe-PVD, to enzymatically generated .O2- . These data show that Fe-PCH, but not Fe-PVD, is capable of catalyzing generation of .OH . Such a process could represent as yet another mechanism of tissue injury at sites of infection with P . aeruginosa. Pathology, 1990 Oct, 22(4), 223 - 6 Serotype and serum sensitivity of Pseudomonas aeruginosa from children with cystic fibrosis: longitudinal studies and typing with monoclonal antibodies; Burke V et al.; Pseudomonas aeruginosa isolated from the respiratory tract of children with cystic fibrosis from Perth, Sydney or Brisbane, were serotyped with polyclonal antisera using the International Antigenic Serotyping System . Predominant strains were untypable among isolates from Brisbane (86% of 50 strains) and Sydney (60% of 50 strains) but 64% of the 408 isolates from Perth were polyagglutinating . The frequency distributions of typable strains showed differences from those reported in clinics in the northern hemisphere, but it appears that these local differences do not affect the emergence of strains with defective lipopolysaccharide antigens . Isolates from patients studied longitudinally showed correlation between duration of colonization, appearance of untypable or polyagglutinating strains and sensitivity to normal serum . Mucoid colonies were found more commonly from patients colonized for at least 12 months but, overall, there was no significant association between serotype and mucoid colonial morphology . Commercial monoclonal antibodies did not react with polyagglutinating or untypable strains and gave identical results to the polyvalent typing system with strains classified as typable. Zentralbl Hyg Umweltmed, 1990 Oct, 190(4), 404 - 11 Antibacterial activity of new synthesized amino-oxides and quaternary ammonium salts; Cupkova V et al.; An amino oxide and three quaternary ammonium salts were investigated for antimicrobial activity . All compounds had low MIC against gram positive bacteria . Despite high MIC the ammonium salts gave good killing of Pseudomonas aeruginosa in time kill experiments . These quaternary ammonium salts may be useful for disinfection as they are easily broken down in the environment. Mol Microbiol, 1990 Oct, 4(10), 1703 - 9 Phage-conversion of cytotoxin production in Pseudomonas aeruginosa; Hayashi T et al.; We isolated a temperate phage which carried the cytotoxin gene (ctx) from a cytotoxin (CTX)-producing Pseudomonas aeruginosa strain, PA158 . The phage, phi CTX, had a head with a hexagonal outline and a contractile tail with tail fibres . The phage genome was a linear double-stranded 35.5 kb DNA with single-stranded cohesive ends (cos) . The attP, cos and ctx genes were all located very close to one another within a 2.3 kb segment on the phage genome in the order given (in the circular form) . phi CTX converted CTX non-producing P . aeruginosa strains into CTX producers . A single copy of phi CTX DNA was integrated at the same site on the host chromosome (attB) in every lysogen, including PA158 . However, the amount of CTX produced in these lysogens varied from strain to strain and was less than that in PA158. FEMS Microbiol Lett, 1990 Oct, 60(1-2), 131 - 5 Uptake and metabolism of methylammonium by Pseudomonas aeruginosa; Jahns T et al.; The mechanism of ammonium uptake was studied in Pseudomonas aeruginosa, measuring the uptake (transport and metabolism) of {14C}methylammonium (MA) . This ammonium analogue was not utilized for growth, but unmetabolized MA was accumulated to intracellular concentrations about 30 times higher than those in the medium . Most of the MA taken up, however, was rapidly metabolized to gamma-N-methylglutamine, which could be removed from the cells by the addition of ammonium . Uptake of MA exhibited distinct optima at pH 7.0 and 35 to 40 degrees C and depended on metabolic energy, as indicated by the inhibitory effect of various metabolic poisons . Growth with ammonium as nitrogen source resulted in the repression of MA uptake, whereas high uptake rates were observed with nitrate or after incubation without nitrogen source . These results suggested that the ammonium/MA uptake system is subject to nitrogen control in P . aeruginosa. J Antimicrob Chemother, 1990 Oct, 26(4), 515 - 24 Synergistic bactericidal interaction of josamycin with human neutrophils in vitro; Labro MT et al.; Josamycin and erythromycin have been compared for their in-vitro interaction with bactericidal killing by human neutrophils . The mechanism of this interaction was studied in two ways . First, the target organisms (Staphylococcus aureus and Pseudomonas aeruginosa) were incubated for 60 min with josamycin, erythromycin or control buffer prior to use in a human polymorphonuclear neutrophil (PMN) killing assay . Second the macrolides were added directly to acellular killing systems mimicking those acting inside the phagolysosome; oxygen-independent systems were obtained from a crude granule extract of PMN and oxygen-dependent systems consisted either of a mixture of xanthine plus xanthine oxidase or of a solution of H2O2 . Whereas josamycin-pretreated P . aeruginosa were twice as sensitive to killing by PMN than were control cells, this was not the case for S . aureus . Both oxidant generating systems were more effective in destroying S . aureus in the presence of josamycin (3 and 30 mg/l) . Erythromycin showed a similar synergy but only with the xanthine plus xanthine oxidase system . This synergy was observed with neither of the O2-independent systems for S . aureus, nor with any acellular system for P . aeruginosa . These data suggest that at least two kinds of mechanism may explain the bactericidal synergy observed between macrolides and PMN . The first (for macrolide-resistant species such as P . aeruginosa) could be due to alterations in the bacteria by the antibiotics, while the second (for macrolide-sensitive species such as S . aureus) could be based upon an as yet unexplained transformation of the molecules by reactive oxygen species into more "toxic" forms . These differences between josamycin and erythromycin could arise from differences in their chemical structure. FEBS Lett, 1990 Oct 1, 271(1-2), 141 - 3 Bacterial ferritin contains 24 haem groups; Kadir FH et al.; Pseudomonas aeruginosa bacterioferritin, also known as cytochrome b1 or cytochrome b557, has been isolated with 9 haems per 24 subunits . Various forms of the protein have been prepared including the completely haem-free protein and the fully haem-loaded protein with 24 haems per 24 subunits . The presence of the core does not significantly affect haem addition or removal . The absorbance ratio of the non-haem-iron-loaded protein, 278 nm:417 nm (oxidised), can be used to estimate the haem loading. Arch Ophthalmol, 1990 Oct, 108(10), 1453 - 9 Experimental Pseudomonas aeruginosa keratitis from extended wear of soft contact lenses; Koch JM et al.; We used a rabbit model to investigate the pathogenesis of soft contact lens-induced bacterial keratitis . Rabbit eyes underwent complete tarsorrhaphy for 7 days either with (group A, n = 14) or without (group B, n = 13) new sterile soft contact lenses . On day 7, an increase in mean corneal thickness (20.3% in group A and 17.2% in group B) was detected . New or rabbit-worn soft contact lenses were then inoculated with 10(7) colony-forming units of Pseudomonas aeruginosa or by 0.1 mL of P aeruginosa suspension . On day 9, conjunctival cultures of all eyes yielded P aeruginosa . Corneal infection developed in 11 of 14 eyes wearing new or worn, contaminated soft contact lenses . Bacterial keratitis did not develop in any of the 13 eyes inoculated with P aeruginosa suspension . Light and electron microscopy of infected eyes showed abundant polymorphonuclear neutrophils destroying the epithelium, basement membrane, and stroma . Few bacteria could be detected and only in the deep stroma . Since bacterial suspension alone caused no inflammation, soft contact lens-wear appears crucial to corneal infection in this model. J Clin Invest, 1990 Oct, 86(4), 1285 - 92 The immunoglobulin G subclass composition of immune complexes in cystic fibrosis . Implications for the pathogenesis of the Pseudomonas lung lesion; Hornick DB et al.; It has been shown that pulmonary macrophage (PM) phagocytosis of Pseudomonas aeruginosa (PA) is inhibited in the presence of serum from cystic fibrosis (CF) patients colonized by Pseudomonas, and that these sera contain high concentrations of IgG2 antibodies . The goal of these studies was to investigate the role that IgG2-containing immune complexes (IC) play in this inhibition of both PM and neutrophil phagocytosis . We found that serum IgG2 concentrations were elevated significantly in CF patients with chronic PA colonization and that in selected sera from CF patients with chronic PA colonization (CF + IC, n = 10), the mean IC level was significantly elevated (2.90 +/- 0.22 mg/dl {SEM}) . IgG2 comprised 74.5% of IgG precipitated in IC from CF + IC sera . An invitro phagocytic assay of {14C}PA uptake using CF + IC whole-sera opsonins confirmed that endocytosis by normal PM and neutrophils was significantly depressed . Removal of IC from CF + IC sera resulted in significantly decreased serum IgG2 concentrations without a significant change in the other subclass concentrations, and enhanced {14C}PA uptake by PM (26.6% uptake increased to 47.3%) and neutrophils (16.9% increased to 52.6%) . Return of the soluble IgG2 IC to the original CF sera supernatants and the positive control sera resulted in return of the inhibitory capacity of the CF + IC sera . We conclude that immune sera from patients with chronic Pseudomonas infections characterized by elevated IgG2 subclass level functions poorly as an opsonin . In these individuals, IgG2 contributes significantly to circulating IC and removal of IC, matched by a simultaneous fall in IgG2, improves bacterial uptake by neutrophil and mononuclear phagocytes . IgG2 antibodies exert antiphagocytic effects by both direct inhibition and the formation of IC. J Bacteriol, 1990 Oct, 172(10), 6162 - 4 Occurrence of D-rhamnan as the common antigen reactive against monoclonal antibody E87 in Pseudomonas aeruginosa IFO 3080 and other strains; Yokota S et al.; S . Sawada and co-workers reported that a monoclonal antibody (MAb), E87, interacted with about 80% of Pseudomonas aeruginosa isolates, and they separated a rhamnose-rich polysaccharide as the probable antigen for MAb E87 from P . aeruginosa IFO 3080 (S . Sawada, T . Kawamura, Y . Masuho, and K . Tomibe, J . Infec . Dis . 152:1290-1299, 1985) . In the present study, the rhamnose-rich polysaccharide was shown to be structurally and immunologically identical to the D-rhamnan of P . aeruginosa IID 1008 (S . Yokota, S . Kaya, S . Sawada, T . Kawamura, Y . Araki, and E . Ito, Eur . J . Biochem . 167:203-209, 1987) . Furthermore, a set of enzymes responsible for the formation of GDP-rhamnose (probably in a D-form) from GDP-D-mannose was found in the 100,000 x g supernatant fractions obtained from all of nine P . aeruginosa strains reactive against MAb E87 . The result strongly supports a possibility that lipopolysaccharides having a D-rhamnan chain widely occur as the common antigen among various P . aeruginosa isolates. J Bacteriol, 1990 Oct, 172(10), 5915 - 23 Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosa; Ostroff RM et al.; Pseudomonas aeruginosa produces two secreted phospholipase C (PLC) enzymes . The expression of both PLCs is regulated by Pi . One of the PLCs is hemolytic, and one is nonhemolytic . Low-stringency hybridization studies suggested that the genes encoding these two PLCs shared DNA homology . This information was used to clone plcN, the gene encoding the 77-kilodalton nonhemolytic PLC, PLC-N . A fragment of plcN was used to mutate the chromosomal copy of plcN by the generation of a gene interruption mutation . This mutant produces 55% less total PLC activity than the wild type, confirming the successful cloning of plcN . plcN was sequenced and encodes a protein which is 40% identical to the hemolytic PLC (PLC-H) . The majority of the homology lies within the NH2 two-thirds of the proteins, while the remaining third of the amino acid sequence of the two proteins shows very little homology . Both PLCs hydrolyze phosphatidylcholine; however, each enzyme has a distinct substrate specificity . PLC-H hydrolyzes sphingomyelin in addition to phosphatidylcholine, whereas PLC-N is active on phosphatidylserine as well as phosphatidylcholine . These studies suggest structure-function relationships between PLC activity and hemolysis. J Bacteriol, 1990 Oct, 172(10), 5540 - 3 Genetic analysis of the Pseudomonas aeruginosa PAO high-affinity branched-chain amino acid transport system by use of plasmids carrying the bra genes; Hoshino T et al.; About 30 mutants of Pseudomonas aeruginosa PAO defective in the high-affinity branched-chain amino acid transport system (LIV-I) were isolated by the selection for resistance to 4-aza-DL-leucine, a toxic leucine analog for LIV-I . All of the mutants were complemented by plasmid pKTH24, harboring the braC gene, which encodes the branched-chain amino acid-binding protein, and the four open reading frames named braD, braE, braF, and braG (T . Hoshino and K . Kose, J . Bacteriol . 172:5531-5539, 1990) . We identified five cistrons corresponding to these bra genes by complementation analysis with various derivatives of pKTH24, confirming that the braD, braE, braF, and braG genes are required for the LIV-I transport system . We also found mutations that seem likely to be mutations in a promoter region for the bra genes and those with polarity in the intercistronic region between braC and braD . Analysis with an omega interposon showed that the bra genes are organized as an operon and are cotranscribed in the order braC-braD-braE-braF-braG from a promoter located in the 5'-flanking region of the braC gene. J Bacteriol, 1990 Oct, 172(10), 5531 - 9 Cloning, nucleotide sequences, and identification of products of the Pseudomonas aeruginosa PAO bra genes, which encode the high-affinity branched-chain amino acid transport system; Hoshino T et al.; A DNA fragment of Pseudomonas aeruginosa PAO containing genes specifying the high-affinity branched-chain amino acid transport system (LIV-I) was isolated . The fragment contained the braC gene, encoding the binding protein for branched-chain amino acids, and the 4-kilobase DNA segment adjacent to 3' of braC . The nucleotide sequence of the 4-kilobase DNA fragment was determined and found to contain four open reading frames, designated braD, braE, braF, and braG . The braD and braE genes specify very hydrophobic proteins of 307 and 417 amino acid residues, respectively . The braD gene product showed extensive homology (67% identical) to the livH gene product, a component required for the Escherichia coli high-affinity branched-chain amino acid transport systems . The braF and braG genes encode proteins of 255 and 233 amino acids, respectively, both containing amino acid sequences typical of proteins with ATP-binding sites . By using a T7 RNA polymerase/promoter system together with plasmids having various deletions in the braDEFG region, the braD, braE, braF, and braG gene products were identified as proteins with apparent Mrs of 25,500, 34,000, 30,000, and 27,000, respectively . These proteins were found among cell membrane proteins on a sodium dodecyl sulfate-polyacrylamide gel stained with Coomassie blue. J Infect Dis, 1990 Oct, 162(4), 914 - 21 Influence of subinhibitory concentrations of cephalosporins on the serum sensitivity of Pseudomonas aeruginosa; Darveau RP et al.; The effect of sublethal concentrations of antibiotics on the serum sensitivity of Pseudomonas aeruginosa was examined . Cefepime, ceftazidime, and imipenem but not amikacin nor ciprofloxacin increased the serum bactericidal activity of pooled normal human serum . Killing was both serum- and antibiotic dose-dependent . Increased sensitivity to the bactericidal action of serum in the presence of cefepime was observed for several different clinical isolates . With the use of C8-deficient sera, the late components of the complement pathway were shown to be essential for bacterial killing . A significant increase in the amount of {125I}C9 based on bacterial mass was observed with bacteria incubated with cefepime compared with non-antibiotic- or amikacin-treated controls . No major change in the amount of type of lipopolysaccharide was observed when cefepime-treated and control bacteria were compared . The data show that cefepime and other cephalosporins at sublethal concentrations increase the complement-mediated bactericidal activity of serum against P . aeruginosa. J Clin Microbiol, 1990 Oct, 28(10), 2178 - 82 Detection of restriction fragment length polymorphisms in clinical isolates and serially passaged Pseudomonas aeruginosa strains; Hjelm LN et al.; An 800-base-pair HindIII-PstI fragment that flanks a hot spot for Tn7 insertion was isolated from the chromosome of Pseudomonas aeruginosa and cloned into pUC12 . The fragment was used to probe XhoI digests of genomic DNA from 18 P . aeruginosa isolates collected from sputum samples of seven cystic fibrosis patients . Only two XhoI restriction fragment length polymorphisms (RFLPs), of 3.7 and 7.7 kilobases (kb), were detected . Isolate WSU3531-1 (3.7-kb XhoI fragment) and WSU3860 (7.7-kb XhoI fragment), while isolated from the same patient, showed different RFLPs . Serial passages of isolate WSU3531-1 demonstrated that this strain was phenotypically stable . In contrast, colony and pigment variants were readily isolated at a frequency of 1% from serial passages of isolate WSU3860 . When XhoI-digested genomic DNA from phenotypic variants of serially passaged WSU3860 were probed with the 800-base-pair HindIII-PstI fragment, the probe hybridized to a 10.4-kb XhoI fragment from three isolates . Restriction analysis of the genomic DNA digested with a variety of restriction enzymes showed that a 2.7-kb insertion occurred in the same region for all three isolates . There appeared to be no correlation between changes in the RFLP and changes in colony morphology. Antimicrob Agents Chemother, 1990 Oct, 34(10), 1889 - 94 Broad-host-range gyrase A gene probe; Robillard NJ; The Escherichia coli gyrase A gene was cloned in the broad-host-range cosmid vector pLA2917 . The resulting plasmid, pNJR3-2, conferred quinolone susceptibility on a gyrA mutant of E . coli . To analyze the expression of this E . coli gene in Pseudomonas aeruginosa, pNJR3-2 or pLA2917 was mobilized via conjugation into P . aeruginosa PAO2 and several well-characterized quinolone-resistant mutants of this strain . The vector pLA2917 did not significantly affect the quinolone susceptibilities of any of the P . aeruginosa strains . However, pNJR3-2 conferred wild-type quinolone susceptibility on P . aeruginosa cfxA (gyrA) mutants and intermediate quinolone susceptibility on cfxA-cfxB double mutants of P . aeruginosa . The quinolone susceptibility of P . aeruginosa PAO2 gyrA+ was unaffected by pNJR3-2 . Also, pNJR3-2 had no significant effect on P . aeruginosa cfxB (permeability) mutants . These results demonstrate that the DNA gyrase A gene from E . coli is expressed in P . aeruginosa and confers dominant susceptibility on gyrA mutants . Thus, pNJR3-2 can be used to detect the quinolone resistance mutations that occur in the gyrase A gene of this organism . pNJR3-2 also appears to discriminate between mutations in gyrA and mutations which alter permeability . This gyrase A probe was used successfully in the analysis of quinolone resistance in clinical isolates of P . aeruginosa. Mol Immunol, 1990 Oct, 27(10), 981 - 93 Identification of functional epitopes of Pseudomonas aeruginosa exotoxin A using synthetic peptides and subclone products; Olson JC et al.; The structure-function relationship of P . aeruginosa exotoxin A (ETA) was examined using synthetic peptides and genetically engineered ETA deletion mutants . Antibodies directed against synthetic peptides have allowed the identification of three ETA epitopes, two within domain I and one within the last 33 amino acids of domain III . In addition two distinct neutralizing determinants have been identified by antibodies directed against subclone products . One was associated with the amino-terminal half of ETA, the proposed receptor binding region . The second was associated with the carboxy-terminal half of ETA, a region previously not associated with receptor-binding . The amino-terminal subclone also offers potential as an ETA vaccine, since it produces a stable, non-enzymatically active product, effective in inducing ETA neutralizing antibodies . Data derived from these studies were used in a re-evaluation of structure-function relationships between ETA and diphtheria toxin. J Bacteriol, 1990 Oct, 172(10), 5544 - 54 A procaryotic regulatory factor with a histone H1-like carboxy-terminal domain: clonal variation of repeats within algP, a gene involved in regulation of mucoidy in Pseudomonas aeruginosa; Deretic V et al.; A novel procaryotic transcriptional regulatory element, AlgP, with a histone H1-like carboxy-terminal domain was identified in Pseudomonas aeruginosa . AlgP is required for transcription of the key biosynthetic gene algD, which is necessary for production of the exopolysaccharide alginate causing mucoidy in P . aeruginosa . Mucoidy is a critical virulence determinant of P . aeruginosa invariably associated with the respiratory infections causing high mortality in cystic fibrosis . Here we show that AlgP and histones H1 both have repeated units of the Lys-Pro-Ala-Ala motif (KPAA) and its variations within their long (over 100 amino acids) carboxy-terminal domains . This region of histone H1 tails has been shown to bind to the linker DNA in eucaryotic chromatin fibers . A synthetic 50-mer peptide consisting of repeats from the AlgP carboxy-terminal domain was found to bind DNA in a mobility shift DNA-binding assay . AlgP is encoded by a gene that contains multiple direct repeats organized as tandem, head-to-tail, 12-base-pair (bp) units overlapping with six highly conserved 75-bp units . The repetitive structure of the algP gene appears to participate in the processes underlying the metastable character of mucoidy in P . aeruginosa . Relatively large DNA rearrangements spanning the region with tandem direct repeats encoding the carboxy-terminal histone H1-like structure of AlgP were detected in several strains upon conversion from the mucoid to the nonmucoid phenotype . The frequency of the detectable algP rearrangements associated with the transition into the nonmucoid state varied from strain to strain and ranged from 0 to 50% . The nonmucoid derivatives with the clearly rearranged chromosomal copy of algP were complemented to mucoidy with plasmids containing algP from P . aeruginosa PAO . When a random collection of mucoid strains, isolated from different cystic fibrosis patients, was analyzed by using polymerase chain reaction, an additional level of strain-dependent sequence variation in algP was observed . Variations in the number of the 12-bp repeats were found; however, they did not appear to influence the mucoid status of the strains examined . Thus, the repeated region of algP appears to be a hot spot for DNA rearrangements and strain-dependent variability. Gene, 1990 Sep 28, 94(1), 83 - 8 Cloning, sequencing, and transcriptional analysis of the recA gene of Pseudomonas cepacia; Nakazawa T et al.; A recombinant plasmid carrying the recA gene of Pseudomonas cepacia complements a recA mutation of Escherichia coli and restores UV and methylmethane sulfonate resistance, as well as recombinational proficiency . The predicted amino acid (aa) sequence of P . cepacia RecA (347 aa; Mr, 37256) is highly homologous to the RecA proteins from Thiobacillus ferrooxidans (74% aa homology), Pseudomonas aeruginosa (72%), E . coli (71%), Anabaena variabilis (61%), and Synechococcus sp . strains PCC7002 (59%) . The transcription of the recA gene in P . cepacia and E . coli, which starts at almost the same site, was enhanced slightly by UV irradiation in the former and markedly in the latter bacteria . An SOS box characteristic to LexA-regulated promoters, along with the -10 and -35 consensus sequences, was found in the 5' upstream region of the P . cepacia recA gene. Biochim Biophys Acta, 1990 Sep 19, 1019(3), 283 - 92 Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli; van de Kamp M et al.; The azurin-encoding azu gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli . A purification procedure was developed to isolate the azurin obtained from the E . coli cells . No differences were observed between azurins isolated from P . aeruginosa and E . coli . A non-reconstitutable azurin-like protein, azurin*, with a spectral ratio (A625/A280) less than 0.01 could be separated from holo-azurin with a spectral ratio of 0.58 (+/- 0.01) . The properties of azurin* were examined by electrophoretic (SDS-PAGE and IEF) and spectroscopic (UV/vis, 1H-NMR, static and dynamic fluorescence) techniques, and compared to the properties of holo-azurin and apo-azurin . Azurin* resembles apo-azurin (same pKa* values of His-35 and His-117, same fluorescence characteristics) . However, it has lost the ability to bind Cu-ions . It is tentatively concluded that azurin* is a chemically modified form of azurin, the modification possibly being due to oxidation of the ligand residue Cys-112 or the formation of a chemical bond between the ligand residues Cys-112 and His-117 . In agreement with previous results from Hutnik and Szabo (Biochemistry (1989) 28, 3923-3934), fluorescence experiments show that the heterogeneous fluorescence decay observed for holo-azurin is not due to the presence of azurin*, but most likely originates from conformational heterogeneity of the holo-azurin. J Biol Chem, 1990 Sep 15, 265(26), 15680 - 4 Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides; Trias J et al.; Protein D2 of Pseudomonas aeruginosa outer membrane is known to facilitate the specific permeation of imipenem (N-formimdoylthienamycin) across this membrane barrier . We have characterized the binding site in the protein D2 channel by studying the competitive inhibition, by various solutes, of imipenem diffusion into the periplasm . We found that basic amino acids, lysine, arginine, histidine, and ornithine, were effective inhibitors . L- and D-lysine were found to be competitive inhibitors with approximate Ki values of 0.6 and 0.3 mM, respectively . Peptides containing L-lysine at the carboxyl terminus, as well as dipeptides containing L-lysine at the amino terminus, were also able to inhibit the transport . Wild type cells transported tripeptide Thr-Ser-Lys into the periplasm three to four times as rapidly as the mutant cells lacking the D2 protein . These results suggest that protein D2 plays a physiologically significant role in the uptake of basic amino acids and peptides containing these amino acids across the outer membrane of P . aeruginosa. Eur J Biochem, 1990 Sep 11, 192(2), 379 - 85 Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa; Bourdenet S et al.; Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa . They possessed both enzymatic activity and residual antigenicity . Their N-terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365-404) . As a result, the polypeptides contained a large portion (13-34 amino acids) of domain Ib linked to the adjacent C-terminal domain III (amino acids 405-613) . The major fragment derived from subtilisin cleavage, at a final yield of 35% (S-fragment; residues 392-613; 24201 Da; pI 4.7) possessed the same level of ADP-ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37-fold higher NAD-glycohydrolase activity . Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP-ribosyltransferase activity of both exotoxin A and the S-fragment, but not the NAD-glycohydrolase activity of the S-fragment . Antibodies against the S-fragment neutralized the ADP-ribosyltransferase activity of exotoxin A . These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392-404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF-2) and show that antibodies recognize the EF-2-binding site but not the NAD(+)-binding site. J Biol Chem, 1990 Sep 5, 265(25), 14728 - 31 Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme; Baur H et al.; Pseudomonas aeruginosa has an anabolic and a catabolic ornithine carbamoyltransferase (OTCase) . In vitro, these homologous enzymes catalyze the same reaction (ornithine + carbamoyl phosphate (CP) in equilibrium citrulline + Pi), yet in vivo they function unidirectionally owing to specific kinetic properties . The catabolic OTC-ase cannot promote the anabolic reaction (citrulline formation) in vivo because of a sigmoidal CP saturation curve and a high CP concentration for half-maximal velocity . The structural basis for this kinetic specialization was examined . The catabolic OTCase lost most of its homotropic cooperativity and gained anabolic activity when an amino acid residue near the CP binding site, Glu-106, was replaced by alanine or glycine . In the anabolic OTCase of Escherichia coli the glutamine residue corresponding to Glu-106 was exchanged for glutamate; however, in this case no CP cooperativity was acquired . Thus, in catabolic OTCase, sequence features in addition to Glu-106 are important for sigmoidal CP saturation, and such a sequence was identified in the C-terminal part . By an in vivo gene fusion technique the 9 C-terminal amino acids of catabolic OTCase were replaced by the homologous 8 amino acids from anabolic OTCase of E . coli; the hybrid enzyme had a markedly reduced homotropic cooperativity . This gene fusion method should be generally useful for directed enzyme evolution. Jpn J Antibiot, 1990 Sep, 43(9), 1614 - 20 {Clinical evaluation of combination therapy with aspoxicillin and ceftazidime for severe infections complicating hematological disorders}; Akasaka K et al.; Clinical effects of a combination therapy using aspoxicillin (ASPC) and ceftazidime (CAZ) were investigated in 88 patients with severe infections which were complicating hematological disorders . ASPC and CAZ were administered intravenously at daily doses of 8 g and 4 to 6 g, respectively, in 2 to 4 divided doses for at least 3 days . The treatment was markedly effective in 20 cases; effective in 31; fairly effective in 4; and ineffective in 33 cases . Seventy-seven patients with whom detailed data were obtained showed an efficacy rate of 63.6% . Bacteria were detected in 9 patients, from whom 10 strains were isolated . The results of bacteriological effects were: 3 strains disappeared, 1 decreased, 3 unchanged, and 3 unclear . The bacteriological eradication rate was 42.9% . Of the detected 10 strains, 3 were identified as Pseudomonas aeruginosa, with 2 of the 3 strains eradicated and 1 decreased . An evaluation of the relationship between clinical efficacies and neutrophil counts before and after the ASPC-CAZ combination therapy showed that the patients with 500/mm3 or higher neutrophil counts before the therapy or those with increased neutrophils after the therapy tended to be more responsive to the therapy . Side effects were observed in 4 patients, but all of them disappeared upon discontinuation of the therapy . The combination therapy with ASPC and CAZ appears to be useful for the treatment of severe infections complicating hematological disorders. Yakugaku Zasshi, 1990 Sep, 110(9), 682 - 7 {Protective activities of a Chinese medicine, hochu-ekki-to, to impairment of hematopoietic organs and to microbial infection}; Ikeda S et al.; Effect of Hochu-ekki-to (HET) on the number of peripheral leukocytes (PL) and their functions in cyclophosphamide (CY)-treated or gamma ray-irradiated mice was investigated . By treatment of mice with anticancer agent CY or gamma ray irradiation, unfavorable side effects usually occurred to impair hematopoietic organs, causing bone marrow disorder . However, it was significantly protected by oral administration of HET (1 g/kg/d) to CY-treated or gamma ray-irradiated mice . The numbers of neutrophils and monocytes in PL were restored to the normal level, and colony-stimulating factor (CSF) was induced in the sera of mice by HET in a dose-dependent manner . The induction of serum CSF reached a peak at 3h after HET administration . Colony-forming unit of bone marrow cells in the spleen adoptively transferred into syngeneic mice, that is defined as CFU-S, was extremely reduced by CY-treatment . However, when HET was orally administered, CFU-S of CY-treated mice was markedly stimulated, suggesting that bone marrow cells were reactivated for further proliferation and mobilization . HET enhanced other leukocyte functions in CY-treated mice; i.e., superoxide produ