Pseudomonas aeruginosa

J Antimicrob Chemother, 2000 Dec, 46(6), 901 - 4
Combined effects of meropenem and aminoglycosides on Pseudomonas aeruginosa in vitro; Nakamura A et al.; To investigate combinations of antibiotics against Pseudomonas aeruginosa, the in vitro effects of combinations of meropenem with each of three aminoglycosides, arbekacin, amikacin and netilmicin, were evaluated using an agar dilution chequerboard technique . The combinations of meropenem and aminoglycosides were effective against almost all P . aeruginosa strains tested, which included meropenem-resistant strains . Increased synergic effects were observed in combinations that included arbekacin or amikacin . None of the combinations had an antagonistic effect . Most of the synergic and additive effects were achieved at clinically relevant concentrations.

J Antimicrob Chemother, 2000 Dec, 46(6), 885 - 93
Influence of the MexA-MexB-oprM multidrug efflux system on expression of the MexC-MexD-oprJ and MexE-MexF-oprN multidrug efflux systems in Pseudomonas aeruginosa; Li XZ et al.; Of the Pseudomonas aeruginosa multidrug efflux systems, MexAB-OprM is expressed in wild-type cells, while MexCD-OprJ is not, and MexEF-OprN shows variable, strain-specific expression . In defined mutant strains, MexCD-OprJ expression increased with decreases in MexAB-OprM and was generally inversely related to MexAB-OprM expression . In so-called wild-type strains expressing MexEF-OprN, MexAB-OprM hyperexpression correlated with a decline in MexEF-OprN expression, while loss of MexAB-OprM was associated with increased expression of MexEF-OprN, also indicative of an inverse correlation between MexAB-OprM and MexEF-OprN expression . Still, the increases in MexCD-OprJ and MexEF-OprN failed to compensate for the loss of MexAB-OprM with respect to antibiotic resistance . Nonetheless, these data suggest that the overall complement of these MDR efflux systems is monitored and that alterations in the level of one efflux system may effect compensatory changes in the levels of the others.

J Clin Microbiol, 2000 Dec, 38(12), 4614 - 5
Evaluation of random amplified polymorphic DNA typing of Pseudomonas aeruginosa; Campbell M et al.; A method for distinguishing among Pseudomonas aeruginosa strains using random amplified polymorphic DNA (RAPD) typing was evaluated for reproducibility and discriminatory power . A total of 200 isolates, blinded in triplicate, were evaluated by RAPD . All 600 samples were typeable; 197 of 200 isolates gave identical results on all three occasions, and 131 distinct RAPD types were identified.

J Clin Microbiol, 2000 Dec, 38(12), 4445 - 52
Simple and inexpensive but highly discriminating method for computer-assisted DNA fingerprinting of Pseudomonas aeruginosa; Al-Samarrai TH et al.; We describe here a method for computer-assisted fingerprinting of Pseudomonas aeruginosa . In this method, DNA is digested with SalI, and bands with molecular sizes of >/=9.7 kb are visually scored after electrophoresis on agarose gels . Pattern scores are entered into a Microsoft Excel database . In scoring, the number of bands within each of a set of molecular size ranges is scored, rather than the absolute molecular size of each band, substantially enhancing the speed and reproducibility of the method, while eliminating the need for using expensive gel scanning equipment and software . Pattern scores are used to generate matrices of genetic distance values, which can be visualized in neighbor-joining trees . The method reliably distinguishes two epidemiologically unrelated isolates in 99.3% of all comparisons . The genetic relationships between isolates observed with the method were consistent with those obtained by analysis of two P . aeruginosa genes, indicating that it provides valid estimates of genetic divergence between isolates . Using the method, respiratory tract isolates from cystic fibrosis patients in Green Lane Hospital in Auckland, New Zealand, were shown to be genetically less diverse than epidemiologically unrelated isolates from other patients . This finding was not due to the existence of clusters of related strains specialized toward colonization of the respiratory tract and thus was indicative of transmission between patients . Analysis of multiple isolates from individual cystic fibrosis patients suggested that up to five separate clusters of genetically related strains may simultaneously be present in a patient . The method described should significantly enhance our ability to investigate the epidemiology of P . aeruginosa.

Biochemistry, 2000 Dec 5, 39(48), 14847 - 64
Interaction of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: conformation of the bound peptide; Campbell AP et al.; The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P . aeruginosa infections . We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein . The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond . The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) . The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135) . These turns have been implicated in cross-reactive antibody recognition . (15)N-edited NMR spectroscopy was used to study the binding of the (15)N-labeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus . The results of these studies are as follows: the trans and cis isomers bind with similar affinity to the Fab, despite their different topologies; both isomers maintain the conformational integrity of their beta-turns when bound; binding leads to the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within regions of the trans and cis backbone that undergo microsecond to millisecond motions . These slow motions may play a role in induced fit binding of the first turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology . More importantly for vaccine design, these motions may also play a role in the development of a broad-spectrum vaccine capable of generating an antibody therapeutic effective against the multiple strains of P . aeruginosa.

Mol Biotechnol, 2000 Sep, 16(1), 5 - 16
Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme; Karmali A et al.; A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase . The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene . The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography . The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method . The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70-80% . The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles . The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography . On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M(r) of 38,000 and 78,000 Dalton, respectively . These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme . Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.

Ann Pharmacother, 2000 Nov, 34(11), 1238 - 42
Safety and tolerability of bolus intravenous colistin in acute respiratory exacerbations in adults with cystic fibrosis; Conway SP et al.; OBJECTIVE: To assess the safety and tolerability of bolus intravenous doses of colistin during acute respiratory exacerbations in adults with cystic fibrosis and chronic Pseudomonas aeruginosa infection . METHODS: Twelve patients with acute exacerbations of cystic fibrosis were enrolled in a Phase I open-label study . On day 1, patients received three doses of colistin 2 mega-units (160 mg), reconstituted in 50 mL of NaCl 0.9%, by infusion over 30 minutes three times daily . On days 2, 3, and 4, the same dose of colistin was administered by bolus injection three times a day over five minutes after reconstitution in 20, 15, and 10 mL of NaCl 0.9%, respectively . The injection was given by a nurse or physician using a hand-held syringe . If the latter dose was tolerated, it was continued for the remaining eight days of the study . If any dose was not tolerated, treatment reverted to the previously tolerated concentration, which was continued throughout the remainder of the study . RESULTS: No serious adverse events occurred during the course of the trial . Patients without total indwelling venous access systems experienced mild to moderate injection pain . There were no clinically significant changes in renal function . CONCLUSIONS: This study indicates that the administration of bolus intravenous colistin as 2 mega-units (160 mg) in 10 mL of NaCl 0.9% three times a day is safe . It is well-tolerated by patients with total indwelling venous access systems.

Appl Environ Microbiol, 2000 Dec, 66(12), 5206 - 12
Influence of infected cell growth state on bacteriophage reactivation levels; Kadavy DR et al.; Reactivation of UV-C-inactivated Pseudomonas aeruginosa bacteriophages D3C3, F116, G101, and UNL-1 was quantified in host cells infected during the exponential phase, during the stationary phase, and after starvation (1 day, 1 and 5 weeks) under conditions designed to detect dark repair and photoreactivation . Our experiments revealed that while the photoreactivation capacity of stationary-phase or starved cells remained about the same as that of exponential-phase cells, in some cases their capacity to support dark repair of UV-inactivated bacteriophages increased over 10-fold . This enhanced reactivation capacity was correlated with the ca . 30-fold-greater UV-C resistance of P . aeruginosa host cells that were in the stationary phase or exposed to starvation conditions prior to irradiation . The dark repair capacity of P . aeruginosa cells that were infected while they were starved for prolonged periods depended on the bacteriophage examined . For bacteriophage D3C3 this dark repair capacity declined with prolonged starvation, while for bacteriophage G101 the dark repair capacity continued to increase when cells were starved for 24 h or 1 week prior to infection . For G101, the reactivation potentials were 16-, 18-, 10-, and 3-fold at starvation intervals of 1 day, 1 week, 5 weeks, and 1 . 5 years, respectively . Exclusive use of exponential-phase cells to quantify bacteriophage reactivation should detect only a fraction of the true phage reactivation potential.

Clin Cardiol, 2000 Nov, 23(11), 808 - 10
Coronary artery stent infection; Dieter RS; This paper aimed to examine the literature for cases of coronary artery stent infection in order to provide comprehensive data to clinicians regarding its prevalence, clinical presentations, and possible treatments . Coronary artery stenting was initially reported in 1987 . Stenting of the coronary arteries is now used in 40-60% of all interventional coronary artery procedures . The understanding of the pathophysiology of coronary artery disease is evolving . It has been suggested that atherosclerosis may be a complication of an infectious etiology . By using a stent to treat coronary artery disease, a foreign body is directly juxtaposed with an area of inflammation . The first reported case of an infected coronary artery stent was in 1993 . Although this is an exceedingly rare event, the associated mortality is alarmingly high . Analysis of the literature reveals a total of four reported cases of coronary artery stent infection . Symptoms of stent infection present days to weeks after the initial coronary intervention . All four patients developed fevers and at least two patients developed postintervention angina . In patients who have had a coronary artery stented, the presence of angina and fevers should make the clinician suspicious for a stent-related infection . Two of the patients had infection with Pseudomonas aeruginosa, which seems to be an unusual organism for a catheter-related infection . Surgical removal of the infected stent and artery complex was performed on nearly all cases . Despite aggressive measures, the majority of patients died . Few data are available on the long-term risk for coronary artery stent infection . In a patient who has undergone coronary artery stent placement, the clinician must be very sensitive to fever, return of angina, and bacteremia . The complication rate at the present time does not warrant the use of prophylactic antibiotics prior to high-risk procedures (e.g., dental procedures) . Furthermore, the low infection rate of coronary artery stents may be a result of the inflammatory nature of atherosclerosis, which may provide a protective benefit against bacterial infection of the stent.

Clin Infect Dis, 2000 Dec, 31(6), 1349 - 56 Epub 2000 Nov 29.
Pseudomonas aeruginosa community-acquired pneumonia in previously healthy adults: case report and review of the literature; Hatchette TF et al.; We report a case of rapidly fatal Pseudomonas aeruginosa community-acquired pneumonia (CAP) in a previously healthy 67-year-old woman . Eleven published case reports of P . aeruginosa CAP in previously healthy adults are reviewed . According to our review, the mean age of affected patients is 45.3 years . Five patients described in the literature were smokers with a mean smoking history of 40 pack-years . The clinical presentation is nonspecific, and although the pneumonia can be rapidly fatal, only 33% of the patients who were reported died . However, mortality may be independent of treatment within the first 36 hours of presentation . Exposure to aerosols of contaminated water is a risk factor for P . aeruginosa CAP in this population . Pseudomonas CAP should be considered in the differential diagnosis for anyone with a smoking history who presents with rapidly progressive pneumonia . We discuss treatment recommendations that are based on evidence in the currently available literature on the subject.

Clin Infect Dis, 2000 Dec, 31(6), 1331 - 7 Epub 2000 Nov 29.
Outbreak of severe Pseudomonas aeruginosa infections caused by a contaminated drain in a whirlpool bathtub; Berrouane YF et al.; During a 14-month period, 7 patients with hematological malignancies acquired serious infections caused by a single strain of multiply resistant Pseudomonas aeruginosa . A case-control study, culture surveys, and pulsed-field gel electrophoresis implicated a whirlpool bathtub on the unit as the reservoir . All case patients and 32% of control patients used this bathtub (P=.003) . The epidemic strain was found only in cultures of samples taken from the bathtub . The drain of the whirlpool bathtub, which was contaminated with the epidemic strain, closed approximately 2.54 cm below the drain's strainer . Water from the faucet, which was not contaminated, became contaminated with P . aeruginosa from the drain when the tub was filled . The design of the drain allowed the epidemic strain to be transmitted to immunocompromised patients who used the whirlpool bathtub . Such tubs are used in many hospitals, and they may be an unrecognized source of nosocomial infections . This potential source of infection could be eliminated by using whirlpool bathtubs with drains that seal at the top.

Microb Pathog, 2000 Dec, 29(6), 345 - 56
Pseudomonas aeruginosa induces apoptosis in human endothelial cells; Valente E et al.; Pseudomonas aeruginosa has been shown to enter into human endothelial cells in vitro . To ascertain the effects of bacterial intracellular (IC) infection, endothelial cells were exposed to PAK and PAO-1 strains for 1 h and treated with gentamicin in culture medium for different periods . P . aeruginosa induced a significant production of superoxide and hydrogen peroxide by endothelial cells . Concentrations of IC bacteria were reduced progressively with time and no viable PAO-1 was detected at 24 h after infection . However, IC infection led to killing of 32.2%+/-2.9 and 51.8%+/-3.5 of the cells infected with PAK and PAO-1, respectively, as determined by the MTT assay . By three criteria (transmission electron microscopy, DNA electrophoresis and reactivity with annexin V) infected cells exhibited features of apoptosis . Treatment of infected cells with anti-oxidants (catalase, tocopherol and N -acetyl-L-cysteine) significantly decreased the percentage of cell death . In contrast, treatment with aminoguanidine, an inhibitor of inducible NO synthase, increased significantly the killing of PAO-1 infected cells . Based on these results we speculate that in response to P . aeruginosa infection, endothelial cells increase the production of reactive oxygen intermediates to eliminate IC pathogens, but cells do not resist the oxidative stress and die by apoptosis .

Microb Pathog, 2000 Dec, 29(6), 329 - 43
Triggering the ExoS regulon of Pseudomonas aeruginosa: A GFP-reporter analysis of exoenzyme (Exo) S, ExoT and ExoU synthesis; Hornef MW et al.; The ExoS regulon of Pseudomonas aeruginosa encodes diverse type III secreted effector proteins which have been shown to exert cytotoxic effects in cell culture experiments . However, little information exists about the environmental conditions and stimuli for upregulation of the ExoS regulon . Translational reporter fusion proteins of exoenzyme (Exo) S, ExoT and ExoU, as well as the type II secreted exotoxin A (ETA) to the green fluorescent protein (GFP), were constructed in order to compare exoprotein production under diverse growth conditions . Reporter protein activity was recorded by FACS-analysis and by conventional and confocal laser scanning microscopy . Low ion concentration induced co-ordinated upregulation of ExoS, ExoT and ExoU with a maximum effect at 37 degrees C . A dose-dependent upregulation was seen with human serum or increasing NaCl concentrations . A type III secretion-negative pcrD mutant of P . aeruginosa showed a weak ExoS response to environmental stimuli, compared with the parental strain, suggesting a negative regulatory mechanism . Co-culture with the mammalian cell lines J774A.1 or HeLa led to rapid upregulation of ExoS, ExoT and ExoU synthesis . These data suggest that the ExoS regulon of P . aeruginosa can be triggered by a variety of environmental signals as well as by cell contact with eukaryotic cells .

Invest Ophthalmol Vis Sci, 2000 Dec, 41(13), 4189 - 94
Membrane-type matrix metalloproteinases in mice intracorneally infected with Pseudomonas aeruginosa; Dong Z et al.; PURPOSE: To establish the presence of membrane-type matrix metalloproteinases (MT-MMPs) in the cornea and their expression in naive and immunized mice intracorneally infected with Pseudomonas aeruginosa . METHODS: Naive (unimmunized) and immunized C57BL/6J mice were infected with P . aeruginosa, and gene expression of MT-MMPs were detected by RT-PCR . Immunoblot analysis and immunostaining were also used to characterize the MT-MMP response in both sets of animals . RESULTS: Expression of MT1-MMP, MT2-MMP, and MT3-MMP (MMP 14, 15, and 16) was detected by RT-PCR and immunoblot analysis . Of the three MT-MMPs detected, MT1-MMP exhibited the greatest expression at protein levels . In general, a bell-shaped curve was obtained for each of the MT-MMPs in naive mice, but all of them showed much less expression in the immunized mice . MT1-MMP was localized in the epithelial tissue of the cornea, whereas MT2-MMP and MT3-MMP were mainly found in the interface between the epithelium and substantia propria . CONCLUSIONS: MT1-MMP was detected and expressed to a greater extent in naive mice than MT2-MMP and MT3-MMP . Peak expression of all three MT-MMPs showed a good correlation with the overall inflammatory response.

Invest Ophthalmol Vis Sci, 2000 Dec, 41(13), 4080 - 4
C57BL/6 mice lacking Muc1 show no ocular surface phenotype; Danjo Y et al.; PURPOSE: To test the hypothesis that a membrane-spanning mucin, Muc1, facilitates the spread of tear film and protects against bacterial adherence . METHODS: Age-matched, Muc1 null mice and wild-type mice of C57BL/6 genetic background were used for comparison . Eyes were examined by slit lamp biomicroscopy with fluorescein solution to assess epithelial damage and tear film stability . Structure of the ocular surface epithelia was examined by light microscopy, scanning and transmission electron microscopy, and wholemount confocal microscopy . Bacterial adherence assay was performed on in vivo corneas with Pseudomonas aeruginosa containing a plasmid encoding green fluorescent protein, followed by wholemount confocal microscopy . Real-time reverse transcription-polymerase chain reaction was performed using Muc4-specific primers to quantitate Muc4 mRNA expression in ocular surface tissues . RESULTS: No differences were found between Muc1 null and control mice in any parameter tested . Ocular surface epithelia of Muc1 null mice of the C57BL/6 strain had a normal appearance of surface microplicae, a well-developed glycocalyx on the apical cell membrane, and a normal appearance of goblet cell mucin packets . There was no convincing evidence that bacterial adherence on the cornea was increased in Muc1 null mice . Muc4 mRNA expression was not upregulated in Muc1 null mice compared with control . No ocular surface infections were observed in Muc1 null mice of the C57BL/6 strain (n = 204), which were housed in the animal facility over a period of 26 months . CONCLUSIONS: Muc1 null mice of C57BL/6 background appeared normal in all respects tested . These data differ from the reported phenotype in the mice of the C57BL/6 x SVJ129 background, which show development of blepharitis and conjunctivitis.

Cornea, 2000 Nov, 19(6), 777 - 81
Pathogenesis and treatment of "sterile" midperipheral corneal infiltrates associated with soft contact lens use; Baum J et al.; PURPOSE: To demonstrate the sterile nature of presumed sterile midperipheral corneal infiltrates associated with soft contact lens (SCL) use and to show that withholding antibiotics or the occasional use of a topical corticosteroid alone may, with strict guidelines, have a role in the treatment of this entity . METHODS: Nine consecutive patients presenting with typical midperipheral corneal infiltrates after SCL wear were seen in the office (O.H.D.) during a 2-year period, 1996-1998 . All patients were initially placed on topical fluorometholone as the only treatment . RESULTS: Eight of the nine patients experienced a rapid relief of symptoms and the infiltrates were noted to be smaller and less dense in 34 days . Therapy was discontinued after 7 days, by which time the lesions had cleared . The ninth patient developed a microbial keratitis from which Pseudomonas aeruginosa was cultured . With appropriate therapy, visual acuity returned to 20/25 . Two different algorithms are offered for the treatment of a putative sterile infiltrate associated with SCL use . CONCLUSION: The use of a topical corticosteroid alone may have a role in the treatment of presumed sterile midperipheral corneal infiltrates associated with SCLs when strict guidelines are followed . Such therapy suggests that the infiltrates are not the result of infection.

Genetika, 2000 Oct, 36(10), 1330 - 9
{Properties of natural interspecific hybrids of transposable phages of Pseudomonas aeruginosa: specific characteristics of phage PL24 transposition}; Mit'kina LN et al.; Properties of natural hybrid transposable phages (TP) of Pseudomonas aeruginosa, including phage PL24 and lysogens for this phage, were studied . PL24 possesses the properties of TP from two previously described groups, B3 and D3112 . Its genome, unlike the genome of D3112, contains many sites susceptible to the SalGI restriction endonuclease and possesses no more than 100 nucleotides of bacterial origin located at the left genome end . However, unlike B3, phage PL24 failed to induce auxotrophic mutants upon integration in the bacterial genome . This phage differed from both B3 and D3112 in sensitivity to chloroform treatment . A more detailed examination of a group containing 25 randomly isolated lysogens for phage PL24 revealed previously unknown processes occurring at early stages of bacterial lysogenization . There are at least two different modes of cell lysogenization with phage PL24 . In the first case, the emerging lysogens contained a single prophage genome located (in each lysogen) at individual sites . In the second case, polylysogenic bacteria appeared, and, after primary integration of a phage genome, replicative transposition occurred at new sites (often accompanied by the appearance of prophage clusters at these sites) . The choice of the mode of lysogenization can be determined both by differences in the physiological state of bacteria and by specific features of phage PL24, which possibly affect the time of repressor accumulation to the concentration sufficient for blocking phage growth or the stability of the lysogenic state.

Braz J Infect Dis, 1999 Jun, 3(3), 97 - 110
Bacterial Pathogens Isolated from Patients with Bloodstream Infections in Latin America, 1997: Frequency of Occurrence and Antimicrobial Susceptibility Patterns from the SENTRY Antimicrobial Surveillance Program; Sader HS et al.; We report the antimicrobial susceptibility of 736 organisms isolated from bloodstream infections in 10 Latin American medical centers during the first six months of 1997 . The data presented here is from the SENTRY Antimicrobial Surveillance Program, a comprehensive surveillance study involving 72 medical centers worldwide . The isolates ivere tested for in in vitro susceptibility to 35 antimicrobial agents by the broth microdilution method . The five most frequently isolated species were (n/%): Staphylococcus aureus (1 65/22.4%), Escherichia coli(118/16.0%), coagulase-negative staphylococci (CoNS - 115/15.6%), Pseudomonas aeruginosa (51/6.9%), Klebsiella spp . (46/ 6.3%) . Susceptibility to oxacillin was 70.9% for S . aureus and only 33.9% for CoNS . Vancomycin was active against all of staphylococci, while teicoplanin was active against 99.4% of S . aureus and only 90.4% of CoNS . The new fluoroquinolones sparfloxacin, gatifloxacin, and trovafloxacin, and the streptogramin, quinupristin/dalfopristin, were very active against these species . Only one vancomycin-resistant enterococcus was detected; however, high-level aminoglycoside resistance rates were common (66.7%) . E . coli and Klebsiella spp . showed low susceptibilities for cefotaxime (90.7% and 41.3%) and for cefoxitin (85.6% and 78.3% respectively), indicating a high frequency of isolates that produce ESBL and/or stably derepressed ampC enzymes . These strains, phenotypically consistent with extended-spectrum beta-lactamase (ESBL) production, were typed using ribotyping and pulsed-field gel electrophoresis . The most active compounds (M IC90 in microg/mL /% susceptibility) against P . aeruginosa were meropenem (2 /94.1%), followed by amikacin (>32 / 86.3%), and piperacillin alone or with tazobactam (128/84.3%) . Ceftazidime and cefepime showed similar activity (70.6% susceptibility) and levofloxacin was the most active fluoroquinolone (MI C50 &lte; 0.5; 76.5% susceptibility) against this gram-negative species . These results show the unique pattern of bloodstream isolates for Latin America and they demonstrate the present utility of several classes of compounds against emerging antimicrobial-resistant species in this region.

Curr Infect Dis Rep, 2000 Dec, 2(6), 490 - 496
Waterborne Nosocomial Infections; Squier C et al.; Waterborne pathogens cause infections in health-care facilities . Despite guidelines addressing these pathogens, outbreaks and pseudo-outbreaks continue to occur . We reviewed recent reports of infections caused by Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Chryseobacterium species, nontuberculous mycobacteria, and Legionella species . Mycobacterium avium complex (MAC) infection in HIV patients has been linked to hospital water distribution systems; molecular subtyping showed that MAC isolates in patients and hospital water were identical . In immunosuppressed patients, Fusarium infection has been linked to the hospital water distribution system; again molecular subtyping showed that isolates from patients and the water supply were identical . Parasites, especially Cryptosporidium, and viruses have also been implicated in nosocomial infection . Transmission occurs via contact, ingestion, aspiration, or aerosolization of potable water, or via the hands of health-care workers . Interventions designed to interrupt transmission of waterborne pathogens have included the use of antimicrobial handwashes, targeted disinfection of the water supply, and, in high-risk populations, restricting the use of tap water.

Curr Infect Dis Rep, 1999 Oct, 1(4), 338 - 346
The Red Menace: Emerging Issues in Antimicrobial Resistance in Gram-Negative Bacilli; Rice LB et al.; Gram-negative bacilli cause more than one third of all nosocomial infections in US hospitals . Despite a surfeit of new and highly potent antimicrobial agents, the problem of resistance in these pathogens continues to increase . Particularly important is the emergence of resistance to the fluoroquinolone and beta-lactam classes of antimicrobial agents . Recent work has confirmed that resistance to fluoroquinolone antibiotics is a complex process that involves mutations in the target enzymes (topoisomerase II and IV), decreased access to the target enzyme resulting from low permeability of the outer membrane (this is primarily important in Pseudomonas aeruginosa), and active efflux from the cell . Resistance to beta-lactam antibiotics, however, is primarily caused by the elaboration of an ever-growing number of beta-lactamases . Our ability to understand the genetic and biochemical underpinnings of these resistance phenotypes will be an important factor in determining the ultimate success of efforts to control their emergence and spread.

J Bacteriol, 2000 Dec, 182(24), 7070 - 4
High-frequency flp recombinase-mediated inversions of the oriC-containing region of the Pseudomonas aeruginosa genome; Barekzi N et al.; The genomes of the two clonally derived Pseudomonas aeruginosa prototypic strains PAO1 and DSM-1707 differ by the presence of a 2 . 19-Mb inversion including oriC . Integration of two Flp recombinase target sites near the rrn operons containing the inversion endpoints in PAO1 led to Flp-catalyzed inversion of the intervening 1.59-Mb fragment, including oriC, at high frequencies (83%), favoring the chromosome configuration found in DSM-1707 . The results indicate that the oriC-containing region of the P . aeruginosa chromosome can readily undergo and tolerate large inversions.

J Bacteriol, 2000 Dec, 182(24), 6999 - 7006
Proteome analysis of the effect of mucoid conversion on global protein expression in Pseudomonas aeruginosa strain PAO1 shows induction of the disulfide bond isomerase, dsbA; Malhotra S et al.; Pseudomonas aeruginosa strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion . The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT {also called AlgU}), which is required for the activation of genes for alginate biosynthesis . In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis . To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its mucA22 derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion . Six proteins that were clearly more abundant in the mucoid strain were observed . The amino termini of such proteins were determined and used to identify the gene products in the genomic database . Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified . This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by mucA mutation . Two other protein spots were also clearly upregulated in the mucA22 background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase) . Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription . The oprF-lacZ fusion showed little difference in levels of expression in the two strains . However, the dsbA-lacZ fusion showed two- to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity . A dsbA-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely, reduction in periplasmic alkaline phosphatase activity, increased sensitivity to dithiothreitol, reduced type IV pilin-mediated twitching motility, and reduced accumulation of extracellular proteases, including elastase . Although efficient secretion of elastase in the dsbA mutant was still demonstrable, the elastase produced appeared to be unstable, possibly as a result of mispaired disulfide bonds . Disruption of dsbA in the mucoid PDO300 background did not affect alginate production . Thus, even though dsbA is coregulated with mucoid conversion, it was not required for alginate production . This suggests that mucA mutation, which deregulates sigma-22, results in a global response that includes other factors in addition to increasing the production of alginate.

J Bacteriol, 2000 Dec, 182(24), 6940 - 9
Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa; Pessi G et al.; Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN) . This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase . The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P . aeruginosa PAO1 . The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR . Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp . Their function was confirmed by transcriptional lacZ fusions . The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1 . Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides . The lux box was recognized by both LasR {activated by N-(oxododecanoyl)-homoserine lactone} and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P . aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P . fluorescens CHA0 . A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression . Without LasR and RhlR, ANR could not activate the hcn promoter . Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone . Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

Intensive Care Med, 2000 Sep, 26(9), 1386 - 9
Endogenous Pseudomonas aeruginosa endophthalmitis: a case report and literature review; Reedy JS et al.; Endogenous endophthalmitis is a vision-threatening condition that results from the hematogenous spread of infection to the eye that originated in a distant primary focus . Although it has long been recognized that bloodborne organisms can infect the eye, endogenous bacterial endophthalmitis is considered a rare entity . We present a unique case of a critically ill patient with a cholangiocarcinoma complicated by ascending cholangitis who developed endogenous Pseudomonas aeruginosa endophthalmitis . An awareness of the risk factors predisposing to endogenous endophthalmitis and a high clinical suspicion are necessary to make an early diagnosis in the intensive care unit . Management involves an aggressive combined medical and surgical approach in an effort to prevent ocular morbidity and vision loss.

Biochemistry, 2000 Nov 28, 39(47), 14504 - 14
Interaction of polyphemusin I and structural analogs with bacterial membranes, lipopolysaccharide, and lipid monolayers; Zhang L et al.; Three structural variants (PV5, PV7, and PV8) of the horseshoe crab cationic antimicrobial peptide polyphemusin I were designed with improved amphipathic profiles . Circular dichroism spectroscopy analysis indicated that in phosphate buffer polyphemusin I, PV7, and PV8 displayed the spectrum of a type II beta-turn-rich structure, but, like polyphemusin I, all three variants adopted a typical beta-sheet structure in an anionic lipid environment . Both polyphemusin I and variants were potent broad spectrum antimicrobials that were clearly bactericidal at their minimal inhibitory concentrations . The variants were moderately less active in vitro but more effective in animal models . Moreover, these variants exhibited delayed bacterial killing, whereas polyphemusin I killed Escherichia coli UB1005 within 5 min at 2.5 microg/mL . All the peptides showed similar abilities to bind to bacterial lipopolysaccharide (LPS) and permeabilize bacterial outer membranes . Consistent with this was the observation that all peptides significantly inhibited cytokine production by LPS-stimulated macrophages and penetrated polyanionic LPS monolayers to similar extents . None of the peptides had affinity for neutral lipids as evident from both tryptophan fluorescence spectroscopy and Langmuir monolayer analysis . As compared to polyphemusin I, all variants showed reduced ability to interact with anionic lipids, and the hemolytic activity of the variants was decreased by 2-4-fold . In contrast, polyphemusin I efficiently depolarized the cytoplasmic membrane of E . coli, as assessed using a membrane potential sensitive fluorescent dye 3,3-dipropylthiacarbocyanine (diSC(3)5) assay, but the variants showed a substantially delayed and decreased depolarizing ability . The coincident assessment of cell viability indicated that depolarization of the bacterial cytoplasmic membrane potential by polyphemusin I occurred prior to lethal damage to cells . Our data suggest that increase of amphipathicity of beta-sheet polyphemusin I generally resulted in variants with decreased activity for membranes . Interestingly, all variants showed an improved ability to protect mice both against infection by Pseudomonas aeruginosa and from endotoxaemia.

J Trauma, 2000 Nov, 49(5), 873 - 8
Locally delivered antibodies combined with systemic antibiotics confer synergistic protection against antibiotic-resistant burn wound infection; Felts AG et al.; BACKGROUND: Nosocomially derived gram-negative infections, particularly from antibiotic-resistant pathogens, are a cause of morbidity in patients with severe burn wounds . METHODS: Locally delivered polyclonal antibodies and systemically infused ceftazidime were combined in a lethal murine burn wound model against a virulent Pseudomonas aeruginosa strain that exhibits intermediate resistance to ceftazidime . RESULTS: Survival was synergistically enhanced in cohorts of burned mice treated both locally (subeschar) with pooled polyclonal human immunoglobulin G (1-mg dose) and intravenously with infused ceftazidime (0.44 mg dose) . Enhancement of survival correlated with reduced bacterial quantitation in local and systemic tissue observed in separate burned cohorts . Burned, infected mice treated prophylactically with either individual treatment at the same dose or a combination of both treatments administered systemically showed no survival enhancement as compared with the untreated control group . CONCLUSION: Treatment of antibiotic-resistant burn wound infections with antibiotics together with locally delivered immunoglobulins may improve antibiotic protective effects against antibiotic-resistant pathogens.

Rev Esp Quimioter, 2000 Sep, 13(3), 281 - 5
{Risk factors and prognostics of nosocomial infectionof surgical wounds in a general hospital}; Martinez B et al.; Surgical infections due to gram-negative bacteria are important because of their high frequency, morbidity and mortality . In order to evaluate the risk factors and prognostics of gram-negative surgical wound infections a group of 50 patients with surgical infections were studied prospectively and consecutively and were compared with another group of 50 patients with similar characteristics but no infection . No significant differences were observed with respect to age between the two groups . Previous surgery, prior surgical infections and use of wide-spectrum antibiotics in the six weeks before the study were significantly associated with the development of surgical wound infections due to gram-negative bacteria . The most isolated bacteria were Pseudomonas aeruginosa (34%), followed by polymicrobial flora (16%) . The factors significantly associated with a poor prognosis were the following: severe underlying disease, a clinically critical situation, previous surgery, arterial hypertension, complications, type of gram-negative bacteria, prior use of wide-spectrum antibiotics in the previous six weeks and older age . No deaths occurred.

J Immunol, 2000 Dec 1, 165(11), 6496 - 503
Intrapulmonary TNF gene therapy reverses sepsis-induced suppression of lung antibacterial host defense; Chen GH et al.; Sepsis syndrome is frequently complicated by the development of nosocomial infections, particularly Gram-negative pneumonia . Although TNF-alpha (TNF) has been shown to mediate many of the pathophysiologic events in sepsis, this cytokine is a critical component of innate immune response within the lung . Therefore, we hypothesized that the transient transgenic expression of TNF within the lung during the postseptic period could augment host immunity against nosocomial pathogens . To test this, mice underwent 26-gauge cecal ligation and puncture (CLP) as a model of abdominal sepsis, followed 24 h later by intratracheal (i.t.) administration of PSEUDOMONAS: aeruginosa . In animals undergoing sham surgery followed by bacterial challenge, PSEUDOMONAS: were nearly completely cleared from the lungs by 24 h . In contrast, mice undergoing CLP were unable to clear P . aeruginosa and rapidly developed bacteremia . Alveolar macrophages (AM) recovered from mice 24 h after CLP produced significantly less TNF ex vivo, as compared with AM from sham animals . Furthermore, the adenoviral mediated transgenic expression of TNF within the lung increased survival in CLP animals challenged with PSEUDOMONAS: from 25% in animals receiving control vector to 91% in animals administered recombinant murine TNF adenoviral vector . Improved survival in recombinant murine TNF adenoviral vector-treated mice was associated with enhanced lung bacterial clearance and proinflammatory cytokine expression, as well as enhanced AM phagocytic activity and cytokine expression when cultured ex vivo . These observations suggest that intrapulmonary immunostimulation with TNF can reverse sepsis-induced impairment in antibacterial host defense.

J Biol Inorg Chem, 2000 Oct, 5(5), 551 - 9
Copper coordination in blue proteins; Gray HB et al.; The spectroscopic and electrochemical properties of blue copper proteins are strikingly different from those of inorganic copper complexes in aqueous solution . Over three decades ago this unusual behavior was ascribed to constrained coordination in the folded protein; consistent with this view, crystal structure determinations of blue proteins have demonstrated that the ligand positions are essentially unchanged on reduction as well as in the apoprotein . Blue copper reduction potentials are tuned to match the particular function of a given protein by exclusion of water from the metal site and strict control of the positions of axial ligands in the folded structure . Extensive experimental work has established that the reorganization energy of a prototypal protein, Pseudomonas aeruginosa azurin, is approximately 0.7 eV, a value that is much lower than those of inorganic copper complexes in aqueous solution . The lowered reorganization energy in the protein, which is attributable to constrained coordination, is critically important for function, since the driving forces for electron transfer often are low (approximately 0.1 eV) between blue copper centers and distant (>10 A) donors and acceptors.

Ann Otolaryngol Chir Cervicofac . 2000 Nov;117(5):291.
{Malignant or necrotizing otitis externa: experience in 22 cases}; Martel J et al.; Malignant or progressive necrotizing otitis extrema is an uncommon but severe infectious condition of the external auditory canal . Over a period of four years, we treated 22 patients: 60% had diabetes (1/4 insulin dependent) and 13% were immunodepressed . The causal germ was Pseudomonas aeruginosa in 87% of cases . The pretherapeutic work-up included a computed tomography scan and a technetium scintigraphy to confirm diagnosis and assess extension . Repeated scintigraphies with gallium were used to follow the course under treatment . Medical treatment was used in most cases (16/22) with parenteral antibiotic therapy using a third-generation cephalosporin (ceftazidime or ceftriaxone) and a fluoroquinolone (ciprofloxacin or ofloxacin) and, if there was no contraindication, hyperbaric oxygen . Surgery is not indicated in malignant otitis externa . We had a 95% cure rate with only 10% recurrence . We reviewed the data in the literature on malignant otitis externa and present the important diagnostic, imaging and therapeutic aspects.

Braz J Infect Dis, 1999 Dec, 3(6), 231 - 237
Evaluation of the Cephalosporins, Cefepime, Cefpirome and Ceftazidime, against Clinical Isolates of Imipenem-Resistant Pseudomonas aeruginosa; Sader HS et al.; The level of resistance to imipenem in our institution has increased significantly in recent years, particularly in isolates of Pseudomonas aeruginosa . At present, 30% to 40% of P . aeruginosa isolated in clinical samples are imipenem resistant . The objective of this study was to test therapeutic alternatives within the class of beta-lactam antibiotics in the treatment of infections caused by imipenem-resistant strains of P . aeruginosa (IRPA) . We tested 160 isolates of IRPA collected consecutively from in patients at Hospital Sao Paulo-UNIFESP (Federal University of Sao Paulo) . Using the E-test method, these isolates were tested for cefepime, cefpirome, and ceftadizime susceptibility or resistance . The E-test was also used to confirm the resistance to imipenem . One group of samples was studied epidemiologically using the pulsed-field gel electrophoresis (PFGE) method to determine how this type of resistance spread in our institution . Cefepime was the most active antibiotic (MIC50, 24 microg/mL; MIC(90), 64 microg/mL) and only 57 (35.6%) of the samples showed cross-resistance between imipenem and this fourth-generation cephalosporin . The number of samples that were highly resistant (MIC(90), >256 microg/mL) to cefepime, ceftazidime or cefpirome was 8 (5.0%), 22 (13.8%) and 38 (23.8%), respectively . Molecular typing revealed the presence of 9 molecular profiles among 26 IRPA samples tested, suggesting that selection of resistant mutants occurred in each patient . However, identical molecular profiles were also found in more than one patient . This study showed that cefepime is the most effective cephalosporin against the IRPA samples isolated in our institution . In addition, we conclude that P.areuginosa resistant strains are selected in each patient, but patient to patient transmission also occurs.

Thorax, 2000 Dec, 55(12), 1033 - 9
Treatment of severe nosocomial pneumonia: a prospective randomised comparison of intravenous ciprofloxacin with imipenem/cilastatin; Torres A et al.; BACKGROUND: A prospective multicentre study was undertaken to compare the efficacy of intravenous ciprofloxacin or imipenem in the treatment of severe nosocomial pneumonia requiring mechanical ventilation . METHODS: Patients with a clinical suspicion of pneumonia were randomised to receive either ciprofloxacin (800-1200 mg/day) or imipenem (2-4 g/day) in doses adjusted for renal function and specimens of the lower respiratory tract were taken . Patients were included in the study when specimens showed significant growth for potentially pathogenic microorganisms in quantitative bacterial cultures (n = 75, ciprofloxacin 41/75 (55%); imipenem 34/75 (45%)) . The clinical and bacteriological success rates were the primary and secondary efficacy variables . An intent-to-treat analysis was performed for all randomised patients who received at least one dose of the study medication (n = 149, ciprofloxacin 72/149 (48%), imipenem 77/149 (52%)) . RESULTS: The success rates were generally good, but neither the clinical success rates (ciprofloxacin, 29/41 (71%), imipenem, 27/34 (79%); 95% CI -10.8 to 28.1; p = 0.435) nor the bacteriological response rate (ciprofloxacin, 20/41 (49%), imipenem, 17/34 (50%); 95% CI -21.5 to 23.9; p = 1.0) were significantly different between the study arms . Pseudomonas aeruginosa was recovered in 26/75 patients (35%) and clinical (ciprofloxacin, 10/14 (71%), imipenem, 8/12 (67%); 95% CI -40.4 to 30.9; p = 1.0) and bacteriological response rates (ciprofloxacin, 7/14 (50%), imipenem, 3/12 (25%), 95% CI -60.9 to 10.9, p = 0.247) were not significantly different in this subgroup of patients . Resistance of Pseudomonas aeruginosa developed in 5/26 cases (19%), 1/14 (7%) to ciprofloxacin and 4/12 (33%) to imipenem (p = 0.147), and the mortality was 12/75 (16%) with no difference between treatment groups (ciprofloxacin, 8/41(24%), imipenem 4/34 (17%); p = 0.362) . The clinical response was evaluable in 109/149 patients (73%) in the intent-to-treat analysis and was successful in 74/109 patients (68%) . The clinical response rates were also not significantly different in the intent-to-treat analysis (ciprofloxacin, 34/52 (65%), imipenem, 40/57 (70%); 95% CI -12.8 to 22.3; p = 0.746) . CONCLUSIONS: Treatment with either ciprofloxacin or imipenem was effective in a selected group of patients with microbiologically confirmed, severe nosocomial pneumonia requiring mechanical ventilation . Although no differences between the study medication could be documented in this trial, smaller differences between treatment arms may have been missed because of sample size limitations.

Infect Immun, 2000 Dec, 68(12), 7100 - 13
The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages; Garrity-Ryan L et al.; Pseudomonas aeruginosa, an important nosocomial pathogen of humans, expresses a type III secretion system that is required for virulence . Previous studies demonstrated that the lung-virulent strain PA103 has the capacity to be either cytotoxic or invasive . Analyses of mutants suggest that PA103 delivers a negative regulator of invasion, or anti-internalization factor, to host cells via a type III secretion system . In this work we show that the type III secreted protein ExoT inhibits the internalization of PA103 by polarized epithelial cells (Madin-Darby canine kidney cells) and J774.1 macrophage-like cells . ExoS, which is closely related to ExoT but has additional ADP-ribosylating activity, can substitute for ExoT as an anti-internalization factor . ExoT contains a signature arginine finger domain found in GTPase-activating proteins . Mutation of the conserved arginine in ExoT diminished its anti-internalization activity and altered its ability to disrupt the actin cytoskeleton . Cell fractionation experiments showed that ExoT is translocated into host cells and that mutation of the arginine finger did not disrupt translocation . In a mouse model of acute pneumonia, PA103DeltaUDeltaT reached the lungs as efficiently as PA103DeltaU but showed reduced colonization of the liver . This finding suggests that the ability to resist internalization may be important for virulence in vivo.

Chest, 2000 Nov, 118(5), 1500 - 3
Hemoptysis following left ventricular aneurysm repair: a misleading clinical sign; Kofidis T et al.; We report on a 66-year-old man with severe hemoptysis following coronary artery bypass grafting and repair of a left ventricular septal defect after acute myocardial infarction . Initial diagnosis was delayed by misleading clinical symptoms and radiologic studies . Due to subfebrile temperature and sputum culture positive for Pseudomonas aeruginosa, he had been treated with antibiotics before reoperation . At reoperation, replacement of all foreign material and reconstruction of the ventricular repair with bovine pericardium resulted in reinfection with the same organism despite prolonged antibiotic therapy after 6 months . Removal of the pericardial tissue with direct suture closure of the ventricles and interposition of omentum led to complete healing of the infection without reoccurrence after 2 years.

Antimicrob Agents Chemother, 2000 Dec, 44(12), 3322 - 7
Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa; Masuda N et al.; To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems . Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities . All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem . Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the beta-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM . Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not . These substrate specificities are distinct from those reported previously.

Antimicrob Agents Chemother, 2000 Dec, 44(12), 3317 - 21
Interactions of bacterial cationic peptide antibiotics with outer and cytoplasmic membranes of Pseudomonas aeruginosa; Zhang L et al.; Polymyxins B and E1 and gramicidin S are bacterium-derived cationic antimicrobial peptides . The polymyxins were more potent than gramicidin S against Pseudomonas aeruginosa, with MICs of 0.125 to 0 . 25 and 8 microg/ml, respectively . These peptides differed in their affinities for binding to lipopolysaccharide, but all were able to permeabilize the outer membrane of wild-type P . aeruginosa PAO1 strain H103, suggesting differences in their mechanisms of self-promoted uptake . Gramicidin S caused rapid depolarization of the bacterial cytoplasmic membrane at concentrations at which no killing was observed within 30 min, whereas, conversely, the concentrations of the polymyxins that resulted in rapid killing resulted in minimal depolarization . These data indicate that the depolarization of the cytoplasmic membrane by these peptides did not correlate with bacterial cell lethality.

Acta Otorhinolaryngol Belg, 2000, 54(3), 367 - 72
Failure of local defense mechanisms in cystic fibrosis; Proesmans M et al.; Cystic fibrosis (CF) is an hereditary disease with pancreatic insufficiency and chronic respiratory tract infections leading to irreversible lung damage as its main features . The typical pathogen for the respiratory disease is Pseudomonas aeruginosa . Although the insight into the pathophysiology and genetics of CF have evolved over the past 10 years, there is not yet a full understanding of the increased susceptibility for respiratory infections, specifically with Pseudomonas aeruginosa . In this overview we elucidate the current knowledge and describe the existing hypothesis on the possible links between the pathophysiological defects and the failure of the local defense mechanism in CF airways.

Plant Physiol, 2000 Nov, 124(3), 1159 - 68
Analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes; Allenbach L et al.; Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle . Two alternative pathways have been described to degrade these fatty acids . One pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme A (CoA) reductase and Delta(3)-Delta(2)-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases . Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also called R-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway . We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the beta-oxidation cycle . Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids.

Otolaryngol Head Neck Surg, 2000 Nov, 123(5), 617 - 23
Controlled multicenter study on chronic suppurative otitis media treated with topical applications of ciprofloxacin 0.2% solution in single-dose containers or combination of polymyxin B, neomycin, and hydrocortisone suspension; Miro N; Otic drops of either ciprofloxacin 0.2% solution (CIP) or a combination of polymyxin B, neomycin, and hydrocortisone suspension (PNH) were administered for 6 to 12 days to patients (14-71 years old) with chronic suppurative otitis media in a randomized, nonblinded, multicenter clinical trial . Two hundred thirty-two enrolled patients were analyzed for efficacy on a "per protocol" basis . The most frequently identified causal agents were Staphylococcus aureus (28% of the patients), Pseudomonas aeruginosa (19%), and Staphylococcus sp (9%) . Clinical success was observed in 91% and 87% of the CIP-and PNH-treated patients, respectively . At 1-month follow-up, 4% of CIP and 6% of PNH patients showed a relapse of otorrhea . Bacteriologic eradication was seen in 89% and 85% of patients in the CIP and PNH groups, respectively . At 1-month follow-up, reinfection or recurrence of infection appeared in 3 patients in the PNH group and in 1 patient in the CIP group . Both treatments were well tolerated . The most frequently reported adverse events were pruritus, stinging, and earache . Audiometric tests did not show changes attributable to study drugs in any but 1 patient in the PNH group . This clinical trial shows that topical 0.2% ciprofloxacin solution in single-dose containers is effective and well tolerated in patients with chronic suppurative otitis media.

Am J Physiol Lung Cell Mol Physiol, 2000 Dec, 279(6), L1199 - 209
Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury; Viget NB et al.; We have previously reported that keratinocyte growth factor (KGF) attenuates alpha-naphthylthiourea-induced lung injury by upregulating alveolar fluid transport . The objective of this study was to determine the effect of KGF pretreatment in Pseudomonas aeruginosa pneumonia . A 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces 4 or 24 h after intratracheal instillation of P . aeruginosa, and the concentration of unlabeled and labeled proteins in the distal air spaces over 1 h was used as an index of net alveolar fluid clearance . Alveolocapillary barrier permeability was evaluated with an intravascular injection of 1 microCi of (131)I-albumin . In early pneumonia, KGF increased lung liquid clearance (LLC) compared with that in nonpretreated animals . In late pneumonia, LLC was significantly reduced in the absence of KGF but increased above the control value with KGF . KGF pretreatment increased the number of polymorphonuclear cells recovered in the bronchoalveolar lavage fluid and decreased bacterial pulmonary translocation . In conclusion, KGF restores normal alveolar epithelial fluid transport during the acute phase of P . aeruginosa pneumonia and LLC in early and late pneumonia . Host response is also improved as shown by the increase in the alveolar cellular response and the decrease in pulmonary translocation of bacteria.

Ter Arkh, 2000, 72(9), 54 - 7
{Some problems of the current therapy of infective endocarditis}; Butkevich OM et al.; AIM: To analyse clinical characteristics of endocarditis for the last 10 years, treatment difficulties and how to overcome them . MATERIAL AND METHODS: 135 patients with infectious endocarditis (IE) were examined according to the routine scheme using modern methods of diagnosis and therapy control: transthoracic and transesophageal echo-CG, test for antibiotics sensitivity of the microflora, etc . Immediate results were assessed in all the patients, some of them were followed up for maximum 5 years . RESULTS: Last decade was marked for growing difficulties in the treatment of IE related to its polyetiology . It can be caused by such therapy-resistant microbes as Staphylococcus aureus, Pseudomonas aeruginosa, anaerobic infection, nosocomial infection, injections of narcotic drugs, etc . CONCLUSION: Current course of IE dictates the necessity of fighting resistant microflora especially in case of nosocomial disease . Recurrences become more frequent . Indications to surgery did not change for the last decade . The best treatment results are achieved after antibacterial treatment of the valve.

Rev Mal Respir, 2000 Aug, 17(3 Pt 2), 758 - 78
{Specific aspects and care of lung involvement in adults with cystic fibrosis}; Pin I et al.; Respiratory impairment is present in almost all adult cystic fibrosis patients and makes the prognosis . Viscous, infected and abundant secretions, inflammation and bronchial oedema, bronchoconstriction and respiratory muscle fatigue lead to airway obstruction, bronchiectasis and respiratory failure . The disease is preferentially located in the upper lobes . Exacerbations of the disease are due to bronchial infections and are often responsible for drops of the respiratory function . Regular spirometric surveillance is fundamental for the prognosis and the assessment of the effects of the treatment . Among adult patients chronic colonisation with mucoid and often multiresistant strains of Pseudomonas Aeruginosa are common . It is treated with i.v . high doses antibiotic courses and nebulized antibiotics between i.v . courses . Respiratory failure may require long term oxygen and non invasive mechanical ventilation . Systemic hypervascularization around the bronchiectasis may lead to moderate to severe hemoptysis, which may require embolization . Pneumothorax are associated with poor prognosis and are treated by pleural drainage and if failure by thoracoscopy.

Rev Mal Respir, 2000 Aug, 17(3 Pt 2), 749 - 57
{Characteristics and specificities of cystic fibrosis in adults: evolutive disease of childhood or recently diagnosed disease?}; Hubert D et al.; We have studied the characteristics of 202 cystic fibrosis adult patients, all with chronic respiratory symptoms, with a median age of 27 yrs (18 to 55 yrs) and a male predominance (56%) . At genetic analysis, delta F508 homozygotes were 41%, delta F508 heterozygotes 42% and 17% had no delta F508 . The respiratory disease was more severe and complications were more frequent in adults: hemoptysis in 14%, pneumothorax in 15%, lung transplantation in 25 patients . Chronic bronchial colonisation with Pseudomonas aeruginosa, in 76% of patients, contributed to making treatments more severe because of antibiotic i.v . courses and nebulised antibiotics . Respiratory function showed a mean FVC of 62 +/- 22% and a mean FEVI of 48 +/- 94% . External pancreatic insufficiency was found in 83%, diabetes in 14% . Intestinal occlusion syndromes were observed in 11% of patients and hepatic cirrhosis in 8% . In spite of the severity of the respiratory disease, theses patients succeeded in social and occupational insertion; 62% were independent, 18% had children and 77% were working or studying . Analysis of the patients according to age at diagnosis showed that, in 38 patients diagnosed after the age of 18 yrs, the respiratory disease was less severe, pancreatic insufficiency and non-respiratory complications were less frequent (34% had pancreatic insufficiency, 5% had diabetes and none had cirrhosis) . This may partly be due to the presence of milder CFTR mutations . In conclusion, cystic fibrosis in adulthood frequently looks like an evolutive form of cystic fibrosis in childhood . Nevertheless, some late diagnosed forms in adults, with better prognosis, have been recently identified.

Rev Mal Respir, 2000 Aug, 17(3 Pt 2), 725 - 32
{Diagnosis and management of cystic fibrosis in children}; Tamalet A et al.; Cystic fibrosis is a genetic disease occurring more frequently in Caucasians . The cystic fibrosis gene, cloned in 1999, codes for the cystic fibrosis transmembrane conductance regulator (CFTR) protein . Dysfunction of this protein leads to the clinical manifestations of cystic fibrosis, mainly lung disease and exocrine pancreas disorders . The pathophysiology of the respiratory component is quite complex, basically related to major and excessive inflammatory processes and to early microbial colonization . Respiratory physical therapy is a key element to management of the respiratory disorder . Antibiotic treatments should be adapted to the bacterial ecology, mainly using antistaphylococcal and antihaemophilus drugs initially, then directed against Pseudomonas aeruginosa . Other drugs including inhaled antiinflammatory drugs are currently in the evaluation stage . In addition, nutritional care and correction of pancreas insufficiency are necessary . The diagnosis of this disease must be made early although systematic neonatal screening is not proposed . Early diagnosis is necessary for improved care and prognosis . Currently, median survival is 29 years . This survival time should probably improve with better understanding of the pathophysiological mechanisms and new therapeutic perspectives.

J Chromatogr B Biomed Sci Appl, 2000 Sep 15, 746(2), 161 - 72
Hydrophobic character of surface regions and total hydrophobicity of four variants of chromosomal class C beta-lactamase from Pseudomonas aeruginosa are identical . Chromatographic comparison of the hydrophobic character of the variants and the effect of focusing buffer composition on the separation of the variants by chromatofocusing with internal and external pH gradients; Walther-Rasmussen J et al.; The hydrophobic character of class C beta-lactamase molecular variants from Pseudomonas aeruginosa was compared by hydrophobic interaction chromatography and reversed-phase liquid chromatography, respectively . Separation of the variants by hydrophobic interaction chromatography was not achieved by modifying salt and pH of mobile phases . Reversed-phase liquid chromatography of the variants resulted in almost identical retention times . The results showed that the hydrophobic character of surface regions as well as total hydrophobicity of the variants are identical . The resolving power of external, internal and gradient chromatofocusing of the variants on strong and weak anion exchangers using low-molecular-mass buffers was compared to that of commercial ampholytes and showed no difference in separation pattern of the variants . Comparisons of variant isoelectric point (pI) values determined by chromatofocusing and isoelectric focusing showed that pI values determined by gradient chromatofocusing were most similar to the pI values determined by isoelectric focusing.

J Bacteriol, 2000 Dec, 182(23), 6550 - 6
Role of Azotobacter vinelandii mucA and mucC gene products in alginate production; Nunez C et al.; Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts . In different bacterial species, the alternative sigma factor sigma(E) regulates the expression of functions related to the extracytoplasmic compartments . In A . vinelandii and Pseudomonas aeruginosa, the sigma(E) factor (AlgU) is essential for alginate production . In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes . In this work, we studied the transcriptional regulation of the A . vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production . Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production.

Clin Infect Dis, 2000 Nov, 31(5), 1131 - 3 Epub 2000 Nov 07.
Cerebrospinal fluid penetration of high doses of intravenous ciprofloxacin in meningitis; Lipman J et al.; Nosocomial meningitis due to gram-negative organisms is a difficult clinical problem to manage because of both antibiotic resistance and poor penetration of many antimicrobials across the blood-brain barrier . Ciprofloxacin has potential in treating this condition when used in high doses . We investigated the plasma and cerebrospinal fluid (CSF) levels of ciprofloxacin in a patient with Pseudomonas aeruginosa meningitis who was treated with 400 mg of intravenous ciprofloxacin every 8 hours . Ciprofloxacin levels in plasma peaked at 10.29 mg/L without resulting in accumulation (8-hour trough levels, <1 mg/L), whereas the CSF level increased to 0.9 mg/L . This CSF level was confirmed to be similar 1 week later . After 1 week of therapy, during which there were no side effects attributable to ciprofloxacin, the organism was eradicated, and there was some clinical improvement . We recommend that 400 mg of intravenous ciprofloxacin every 8 hours be considered for treatment of difficult-to-treat gram-negative bacillary meningitis.

Clin Infect Dis, 2000 Nov, 31(5), 1119 - 25 Epub 2000 Nov 06.
Hospital outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-1, a novel transferable metallo-beta-lactamase; Cornaglia G et al.; A total of 8 Pseudomonas aeruginosa isolates was collected from 7 different patients in different wards of the University Hospital of Verona, Italy, from February 1997 to February 1998 . The high level of resistance to carbapenems (imipenem minimum inhibitory concentration was always >128 microg/mL) and other broad-spectrum beta-lactams and the rate of imipenem hydrolysis and its inhibition by ethylenediamine-tetra-acetic acid were all suggestive of production of a carbapenem-hydrolyzing metallo-beta-lactamase . A specific DNA probe derived from the recently cloned bla(VIM-1) gene hybridized to all the isolates . A genomic DNA fingerprinting profile revealed clonal relatedness for 7 of 8 isolates . A description of this hospital outbreak is reported, the occurrence of which confirms that proliferation of metallo-beta-lactamase-producing strains multiply resistant to beta-lactams is already a reality outside Japan . These findings emphasize the need for early recognition of similar isolates.

J Hosp Infect, 2000 Nov, 46(3), 203 - 9
Assessment of in-vitro efficacy of 1% Virkon against bacteria, fungi, viruses and spores by means of AFNOR guidelines; Hernndez A et al.; Peroxygenic acid, under the brand name Virkon, has unleashed great debate following contradictory reports of its efficacy and spectrum of activity . The aim of this study was to test the biocidal activity of the compound against 10 different micro-organisms, following standard in-vitro test procedures . Bactericidal, fungicidal and sporicidal activities were determined using quantitative suspension and germ carrier tests and virucidal activity was assessed using a simple dilution suspension test, following the Association Francaise de Normalisation (AFNOR) guidelines . One percent Virkon demonstrated bactericidal activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus hirae and Mycobacterium smegmatis in the suspension test and against P . aeruginosa, E . coli, S . aureus and E . hirae in the carrier test . One percent Virkon showed virucidal activity against poliovirus in the suspension test . However, this concentration did not comply with sporicidal and fungicidal activity guidelines . In conclusion, 1% Virkon is effective only against vegetative bacteria, yeasts and viruses, and should therefore be considered a low-level disinfectant .

J Leukoc Biol, 2000 Nov, 68(5), 700 - 6
Losartan, a selective inhibitor of subtype AT1 receptors for angiotensin II, inhibits neutrophil recruitment in the lung triggered by fMLP; Raiden S et al.; We have shown that losartan, a selective inhibitor of AT1 receptors for angiotensin II (AII), inhibits the binding of {3H}fMLP to neutrophil receptors (FPR) . Here, we analyze, in Wistar rats, the effect of losartan on neutrophil recruitment in the lung triggered by fMLP . We found that i.v . infusion of losartan (0.4-20.0 microg/kg/min) inhibits neutrophil recruitment induced by i.t . instillation of fMLP, without affecting the responses induced by other stimuli, such as aggregated human IgG (aIgG), precipitating immune complexes (IC), or zymosan . Histological evaluation of lungs as well as the analysis of lung hemorrhage indices showed that losartan prevents tissue injury partially in fMLP-challenged rats . We also analyzed the effect of losartan on lung-neutrophil recruitment triggered by i.t . instillation of Pseudomonas aeruginosa . Not only was there a marked decrease in neutrophil recruitment but also a significant increase in the survival of rats instillated with Pseudomonas aeruginosa, as a consequence of losartan treatment . Our results support the notion that losartan may be useful in the treatment of certain lung inflammatory disorders associated with bacterial infectious diseases.

Respiration, 2000, 67(5), 477 - 90
Early detection of lung disease and its association with the nutritional status, genetic background and life events in patients with cystic fibrosis; Kraemer R et al.; Progression of lung disease is the most prominent cause of morbidity and death in patients with cystic fibrosis (CF), but the severity of lung disease and the rate of lung function decline are highly variable . An attempt was made to define accurate estimates of disease progression in these patients early diagnosed and prospectively evaluated until 10 years of age . The primary question to ask was whether functional abnormalities detected already in infancy are associated with functional derangements later on in life, and may be useful as parameters of prognostic value . Early diagnosis of CF can best be achieved by screening of mutation by new techniques (buccal cell brushing) in infants, even when the sweat test or accurate blood sampling is not available . Moreover, in infants lung function can be assessed by infant whole-body plethysmography enabling the study of the interrelationship with delayed weight gain and growth retardation, as well as the associations with the most common disease-causing mutations . Out of a cohort of 80 infants (39 males, 41 females) with CF a follow-up study was started with 50 CF infants diagnosed during infancy (mean age 4.6 +/- 4.0 months; range 0.1-12.7 months) and prospectively evaluated at 6-month intervals during the first 2 years of life . Moreover, in 32 CF children out of this cohort, follow-up was continued until 10 years of age . Differences were encountered with respect to the different events occurring during the first years of life, especially the onset of chronic colonization with Pseudomonas aeruginosa . The association between infant lung function and specific mutations (DeltaF508 homozygotes, frameshift DeltaF508/3905insT compound heterozygotes and nonsense DeltaF508/R553X compound heterozygotes) furthermore revealed that differences in lung function within the genetic groups are mainly related to the degree of pulmonary hyperinflation . Pulmonary hyperinflation was also associated with the degree of impaired nutritional status . An association between impaired gas exchange characteristics at 10 years of age and the degree of pulmonary hyperinflation during infancy finally demonstrates that by early mutation screening, lung function testing and assessment of the nutritional status predictors of disease progression later on in life can be defined . Therefore, preventive therapeutic measures should primarily be based on such prognostic factors .

Mol Microbiol, 2000 Oct, 38(2), 213 - 31
The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage; Nakayama K et al.; Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins . The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one . As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage . In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P . aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages . The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage . This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages . The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins . A systematic polymerase chain reaction (PCR) analysis of P . aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.

Lett Appl Microbiol, 2000 Oct, 31(4), 313 - 8
Monoclonal antibody detection of naphthalene dioxygenase from Pseudomonas aeruginosa 2NR; Civilini M et al.; A monoclonal antibody, designated mAb alpha(CT), was generated against a peptide of the ISP(NAP) alpha-subunit of the naphthalene dioxygenase (NDO) enzyme of Pseudomonas aeruginosa . Since NDO expression is induced by aromatic hydrocarbons, its detection is important as a tool for environmental biomonitoring . This antibody is highly specific and works well both in an indirect ELISA assay and Western Blot analysis, allowing the detection of Pseudomonas spp . expressing the NDO inducible enzyme . The detection threshold for the ELISA assay developed in this work was 10(4) colony forming units (cfu) per ml . Thus, this mAb could represent a powerful tool to test for pollutants in soil, groundwater, and other natural environments.

Microbiology, 2000 Nov, 146 ( Pt 11), 2803 - 14
Involvement of the rml locus in core oligosaccharide and O polysaccharide assembly in Pseudomonas aeruginosa; Rahim R et al.; L-Rhamnose (L-Rha) is a component of the lipopolysaccharide (LPS) core, several O antigen polysaccharides, and the cell surface surfactant rhamnolipid of Pseudomonas aeruginosa . In this study, four contiguous genes (rmlBDAC) responsible for the synthesis of dTDP-L-Rha in P . aeruginosa have been cloned and characterized . Non-polar chromosomal rmlC mutants were generated in P . aeruginosa strains PAO1 (serotype O5) and PAK (serotype O6) and LPS extracted from the mutants was analysed by SDS-PAGE and Western immunoblotting . rmlC mutants of both serotype O5 and serotype O6 synthesized a truncated core region which was unable to act as an attachment point for either A-band or B-band O antigen . A rmd rmlC PAO1 double mutant (deficient in biosynthesis of both D-Rha and L-Rha) was constructed to facilitate structural analysis of the mutant core region . This strain has an incomplete core oligosaccharide region and does not produce A-band O antigen . These results provide the genetic and structural evidence that L-Rha is the receptor on the P . aeruginosa LPS core for the attachment of O polysaccharides . This is the first report of a genetically defined mutation that affects the synthesis of a single sugar in the core oligosaccharide region of P . aeruginosa LPS, and provides further insight into the mechanisms of LPS biosynthesis and assembly in this bacterium.

Pediatr Pulmonol, 2000 Nov, 30(5), 413 - 24
Mouse models of chronic lung infection with Pseudomonas aeruginosa: models for the study of cystic fibrosis; Stotland PK et al.; The discovery of the CFTR gene in 1989 has lead to rapid progress in understanding the molecular basis of cystic fibrosis (CF) and the biological properties of the cystic fibrosis transmembrane conductance regulator (CFTR) protein . However, more than 10 years later, recurrent lung infections with Pseudomonas aeruginosa, which lead to chronic lung disease and eventual respiratory failure, remain the major cause of morbidity and mortality among CF patients . A distinguishing feature of lung disease in CF is an exaggerated and persistent inflammatory response, characterized by the accumulation of excessive numbers of neutrophils and dysregulated cytokine production . The events leading to the establishment of lung infection with P . aeruginosa, especially the inflammatory and immunological events, and the relation between the CF defect and infection, remain largely undefined . Progress in this area has been hampered by the lack of a suitable animal model . An exciting achievement in the past few years has been the development of a number of variants of CFTR-deficient mice which exhibit defective cAMP-mediated Cl(-) conductance and have a range of clinical phenotypes from mild to severe . In parallel, a model of chronic P . aeruginosa lung infection has been established in genetically and immunologically well-defined inbred mouse strains which differ in susceptibility to this infection in the lung . BALB/c mice are resistant, while DBA/2 mice are extremely susceptible, with high mortality within 3 days of infection . C57BL/6 and A/J mice are relatively susceptible and experience low mortality . Furthermore, the bacterial load correlates with the magnitude and quality of the inflammatory response in the infected lungs of BALB/c and C57BL/6 mice . Although results of infection studies in CFTR-deficient mice have been variable, C57BL/6-Cftr(m1UNC)/Cftr(m1UNC) knockout mice compared to littermate control mice are highly susceptible to chronic P . aeruginosa infection in the lung . The availability of CFTR knockout mice and non-CF inbred mice differing in susceptibility to chronic P . aeruginosa infection offers useful tools for progress in understanding the genesis of chronic P . aeruginosa infection and the ensuing inflammation in the CF lung, as well as the relation between the CF defect and infection . Information generated from these studies will provide the rationale for the development of novel immunomodulatory measures capable of ameliorating or modulating the chronic inflammation associated with CF lung disease .

FEMS Immunol Med Microbiol, 2000 Nov, 29(3), 227 - 32
The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa; Schultz MJ et al.; To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P . aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA . P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05) . P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody . The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.

FEMS Microbiol Lett, 2000 Nov 15, 192(2), 205 - 10
Respiratory pathways of Rhodobacter sphaeroides 2.4.1(T): identification and characterization of genes encoding quinol oxidases; Mouncey NJ et al.; Rhodobacter sphaeroides 2.4.1(T) respires aerobically via a branched respiratory chain consisting of both cytochrome c oxidases and quinol oxidases . Here, genes from chromosome II encoding two distinct quinol oxidases have been characterized . The qoxBA genes encode a putative heme-copper quinol oxidase, whereas the qxtAB genes encode a quinol oxidase homologous to the cyanide-insensitive oxidase of Pseudomonas aeruginosa . No phenotype was observed for mutations in either oxidase in the wild-type background . A strain containing a qxtA mutation in a cytochrome bc(1) complex mutant background was unable to grow aerobically . No role was found for the Qox oxidase, nor was a qoxB::lacZ transcriptional fusion expressed under a variety of conditions . These are the first molecular studies to characterize the quinol oxidases of R . sphaeroides 2.4.1(T).

Harefuah, 2000 Oct, 139(7-8), 267 - 9, 327
{Corneal infection in wearers of contact lenses: causes, effect on visual acuity and prevention}; Domniz Y et al.; This is a 5-year retrospective survey of corneal infection in wearers of optical contact lenses (OCL) . 23 of the 61 patients (38%; Hasharon Hospital) with positive cultures wore OCL . Visual acuity improved in 15 (65%), no change was noted in 4 (17.5%) and there was deterioration in 4 (17.5%), as compare with status on admission . Pseudomonas aeruginosa was the most common cause of infections among OCL wearers . The improvement in visual acuity expected due to wearing OCL was affected by infections . Those after Staphylococcus albus infections had the highest rate (100%) of improvement in visual acuity and after Ps . aeruginosa the lowest rate (57.2%) of improvement, as well as the highest rate of deterioration (42.8%) found following recovery . OCL wearers are at higher risk for damage to visual acuity following corneal infection, and highly virulent infections in OCL wearers are responsible for a high risk of damage to visual acuity.

J Antimicrob Chemother, 2000 Nov, 46(5), 733 - 9
A combined in vivo pharmacokinetic-in vitro pharmacodynamic approach to simulate target site pharmacodynamics of antibiotics in humans; Delacher S et al.; We describe a new approach to quantify in vivo anti-infective activity by simulating effect site pharmacokinetics of antibiotics in vitro . This approach is based on (i) the in vivo measurement of interstitial drug pharmacokinetics (PK) at the target site and (ii) a subsequent pharmacodynamic (PD) simulation of the time versus drug concentration profile in an in vitro setting . To demonstrate the feasibility of this approach, individual time-concentration profiles of ciprofloxacin were measured in the interstitial space fluid of eight healthy volunteers by microdialysis following iv administration of 200 mg . Thereafter, different isolates of Pseudomonas aeruginosa were exposed in vitro to the interstitial ciprofloxacin concentration profile obtained from in vivo experiments . This led to a 1- to 3-log10 decrease in the number of viable organisms after 8 h . Significant correlations were observed between the maximal bactericidal effect and several PK surrogate parameters, notably the AUC/MIC ratio (P: = 0.0005), the C:max/MIC ratio (P: = 0.006) and the time > MIC (P: = 0.02) . Furthermore, the data were analysed with an integrated PK-PD model allowing a much more detailed evaluation of the data than using MIC . The model employed an E:max relationship to link unbound ciprofloxacin concentration to bacterial kill rate . In conclusion, our experiments show that therapeutic success and failure in antimicrobial therapy may be explained by pharmacokinetic variability at the target site . Therefore, the in vivo PK-in vitro PD approach presented in our study may provide valuable guidance for drug and dose selection of antimicrobial agents.

Pulm Pharmacol Ther, 2000, 13(6), 293 - 9
CGS 21680, dibutyryl cyclic AMP and rolipram attenuate the pro-inflammatory interactions of the Pseudomonas aeruginosa -derived pigment, 1-hydroxyphenazine, with human neutrophils; Ramafi G et al.; The effects of the intracellular adenosine 3':5' cyclic monophosphate (cAMP)-elevating agents, CGS 21680 (0.01- 1 microM) and rolipram (0.01-1 microM), as well as those of dibutyryl cAMP (0 . 05-4 mM) on the pro-inflammatory interactions of the P . aeruginosa -derived pigment, 1-hydroxyphenazine (1-hp, 3.1 and 12.5 microM), with human neutrophils have been investigated in vitro . Ca(2+)fluxes in FMLP-activated neutrophils were measured using a fura-2/AM spectrofluorimetric procedure, while a colourimetric method was used to measure release of the primary granule enzyme, elastase, from the cells . Treatment with 1-hp resulted in delayed clearance of Ca(2+)from the cytosol of N -formyl- L -methionyl- L -leucyl- L -phenylalanine (FMLP, 1 microM)-activated neutrophils and increased release of elastase . All 3 test agents caused dose-related antagonism of 1-hp-mediated potentiation of elastase release from activated neutrophils, which was associated with restoration of Ca(2+)homeostasis . These observations demonstrate the potential of cAMP-elevating agents, acting on Ca(2+)clearance mechanisms in activated neutrophils, to attenuate the potentially harmful pro-inflammatory effects of 1-hp .

Schweiz Med Wochenschr, 2000 Sep 30, 130(39), 1366 - 72
{Inhaled colistin in cystic fibrosis}; Tamm M et al.; The clinical course of cystic fibrosis (CF) is characterised by chronic bronchial infection with Pseudomonas aeruginosa . Therapy with inhaled aminoglycosides was introduced to decrease the rate of infectious exacerbations and to delay pulmonary progression . However, development of resistance to aminoglycosides is frequent . Few investigations are available into the resistance profile under treatment with colistin . Antibiotic resistance to colistin was analysed in 44 adult CF patients treated with inhaled colistin . Resistance to aminoglycosides was observed in 86% of cases (38/44) before therapy and decreased to 43% (19/44) under treatment with colistin . Five patients (11%) developed polymyxin resistance . After cessation of therapy pseudomonas became sensitive to polymyxin within a few months and enabled colistin to be reintroduced . In addition, we performed a pilot study analysing the effect of inhaled colistin on the growth of pseudomonas . The number of Pseudomonas aeruginosa decreased from 16.7 million (CFU) bacteria per ml sputum to 2.9 million under therapy with colistin . There was a more than tenfold increase in bacterial counts after inhaled colistin was stopped . Genotyping revealed no change in the type of pseudomonas strains . CONCLUSION: Development of resistance to polymyxin is not rare under long-term treatment with inhaled colistin and requires temporary interruption of therapy . Sputum cultures should therefore be tested regularly for polymyxin resistance in patients treated with inhaled colistin.

Am Fam Physician, 2000 Oct 15, 62(8), 1870 - 6
Topical fluoroquinolones for eye and ear; Morden NE et al.; Topical fluoroquinolones are now available for use in the eye and ear . Their broad spectrum of activity includes the common eye and ear pathogens Staphylococcus aureus and Pseudomonas aeruginosa . For the treatment of acute otitis externa, these agents are as effective as previously available otic preparations . For the treatment of otitis media with tympanic membrane perforation, topical fluoroquinolones are effective and safe . These preparations are approved for use in children, and lack of ototoxicity permits prolonged administration when necessary . Topical fluoroquinolones are not appropriate for the treatment of uncomplicated conjunctivitis where narrower spectrum agents suffice; they represent a simplified regimen for the treatment of bacterial keratitis (corneal ulcers) . When administered topically, fluoroquinolones are well tolerated and offer convenient dosing schedules . Currently, bacterial resistance appears limited.

Crit Care, 2000, 4(4), 255 - 61 Epub 2000 Jul 07.
Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid 'real-time' polymerase chain reaction; Pirnay JP et al.; STATEMENT OF FINDINGS: We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples . This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction . The time from sample collection to result was less than 1h . RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.

Appl Environ Microbiol, 2000 Nov, 66(11), 4735 - 41
Ingested blood contributes to the specificity of the symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech; Indergand S et al.; Hirudo medicinalis, the medicinal leech, usually carries in its digestive tract a pure culture of Aeromonas veronii bv . sobria . Such specificity is unusual for digestive tracts that are normally colonized by a complex microbial consortium . Important questions for the symbiotic interaction and for the medical application after microvascular surgery are whether other bacteria can proliferate or at least persist in the digestive tract of H . medicinalis and what factors contribute to the reported specificity . Using a colonization assay, we were able to compare experimentally the ability of clinical isolates and of a symbiotic strain to colonize H . medicinalis . The symbiotic A . veronii bv . sobria strain proliferated well and persisted for at least 7 days inside the digestive tract . In contrast, the proliferation of Pseudomonas aeruginosa and Staphylococcus aureus was inhibited inside the animal compared to growth in the in vitro control, indicating that the ingested blood was modified within the digestive tract . However, both strains were able to persist in the digestive tract for at least 7 days . For an Escherichia coli strain, the viable counts decreased approximately 1, 000-fold within 42 h . The decrease of viable E . coli could be prevented by interfering with the activation of the membrane-attack complex of the complement system that is present in blood . This suggests that the membrane-attack complex remained active inside H . medicinalis and prevented the proliferation of sensitive bacteria . Thus, antimicrobial properties of the ingested vertebrate blood contribute to the specificity of the A . veronii-H . medicinalis symbiosis, in addition to modifications of the blood inside the digestive tract of H . medicinalis.

Pediatr Infect Dis J, 2000 Oct, 19(10), 959 - 63
Pseudomonas aeruginosa bacteremia in children: analysis of trends in prevalence, antibiotic resistance and prognostic factors; Grisaru-Soen G et al.; OBJECTIVE: To determine the factors predisposing to Pseudomonas aeruginosa bacteremia as well as the prevalence, source of infection, outcome and prognostic factors in pediatric patients . METHODS: Retrospective review of pediatric patients with P . aeruginosa bacteremia, at a large tertiary care hospital during a 6.5-year period . RESULTS: Seventy patients with P . aeruginosa bacteremia were identified . The annual rate of P . aeruginosa bacteremia remained unchanged during the study period . Antibiotic susceptibility remained unchanged except for two patients with extensive burns who developed resistant strains . Underlying diseases were malignancy (50%), prematurity (6%), burns (7%) and others (37%) . The overall mortality associated with P . aeruginosa bacteremia was 20% . The fatality rate was higher among the young infants (compared with older children) and those who received previous antibiotic therapy (P = 0.02) . Mortality rate was higher in nosocomial than in community-acquired infections (25% compared with 11.5%) . The mortality rate of low birth weight and burns patients was significantly higher when compared with oncology patients or other patients, 75 and 40% compared with 11 and 19%, P = 0.01 . Multiple regression analysis revealed a correlation only between the underlying disease and mortality (P = 0.02) . In the oncology patients the only significant risk factor for mortality was absolute neutrophil count < or =0.1 x 10(9)/l (P = 0.06) . CONCLUSION: P . aeruginosa bacteremia, although apparently not increasing in incidence and antibiotic resistance, is still a common serious complication in immunocompromised children with a high mortality rate . We conclude that the underlying disease is the main determinant of the clinical outcome.

Am J Kidney Dis, 2000 Nov, 36(5), 1009 - 13
Analysis of microbiological trends in peritoneal dialysis-related peritonitis from 1991 to 1998; Zelenitsky S et al.; The microbial cause of peritoneal dialysis-related peritonitis is an important determinant of clinical outcome and the basis of widely used treatment guidelines . Five hundred forty-six cases of peritonitis in 374 patients from 1991 to 1998 were analyzed . The rate of peritonitis declined significantly from 1.37 episodes/patient-year in 1991 to 0.55 episode/patient-year in 1998 (P = 0.02) . The rate of Gram-positive peritonitis decreased significantly from 0.75 to 0.28 episode/patient-year during the same period (P = 0.02) . Conversely, the occurrence of Gram-negative peritonitis remained constant at approximately 0.16 episode/patient-year (P = 0.28) . Staphylococcus epidermidis and Staphylococcus aureus were the most common causes of peritonitis, isolated in 27.8% and 19.3% of the culture-positive cases, respectively . A distinct decrease in peritonitis caused by S epidermidis was observed, with 0.40 episode/patient-year in 1991 compared with 0.11 to 0.20 episode/patient-year during subsequent years . The rate of infections caused by S aureus decreased significantly over time from a high of 0.21 episode/patient-year in 1992 to a low of 0.04 episode/patient-year in 1998 (P = 0.01) . Pseudomonas aeruginosa, Escherichia coli, and KLEBSIELLA: species were the most common causes of Gram-negative peritonitis, identified in 7.1%, 6.8%, and 5.2% of culture-positive cases, respectively . The most dramatic increase in antibiotic resistance was seen among S epidermidis . From 1991 and 1992 to 1997 and 1998, resistance to ciprofloxacin increased from 5.4% to 47.8% (P = 0.003), and resistance to methicillin increased from 18.9% to 73.9% (P = 0.03) . Our study showed significant trends in the causative pathogens of peritoneal dialysis-related peritonitis and dramatic increases in antibiotic resistance . These data support further study and warrant reevaluation of current treatment practices.

Chemotherapy, 2000 Nov-Dec, 46(6), 383 - 9
Comparison of the bactericidal activity of trovafloxacin and ciprofloxacin, alone and in combination with cefepime, against Pseudomonas aeruginosa; McNabb J et al.; BACKGROUND: Although ciprofloxacin exhibits more intense microbiological activity against Pseudomonas aeruginosa than does trovafloxacin, the clinical relevance of this observation remains questionable, particularly when the agents are combined with another antipseudomonal agent . METHODS: To evaluate this further, we conducted a four-way crossover trial to compare the bactericidal activities of ciprofloxacin and trovafloxacin, alone and in combination with cefepime, against three clinical isolates of P . aeruginosa . Healthy subjects received the following regimens, dosed to steady state: trovafloxacin 300 mg/24 h; ciprofloxacin 400 mg/12 h; trovafloxacin 300 mg/24 h plus cefepime 2 g/12 h, and ciprofloxacin 400 mg/12 h plus cefepime 2 g/12 h . Serum bactericidal titers were performed with each regimen . RESULTS: As monotherapy, the area under the bactericidal curve for ciprofloxacin exceeded that of trovafloxacin for all isolates . No significant difference in the overall degree of bactericidal activity was noted for two of three P . aeruginosa isolates for the combination regimens . Additionally, both combination regimens provided bactericidal activity for 100% of the dosing interval for all isolates . CONCLUSION: These results indicate that, while in vitro differences exist among these quinolones for P . aeruginosa, when a fluoroquinolone is combined with a beta-lactam, this is likely to be of little clinical significance .

Acta Crystallogr D Biol Crystallogr, 2000 Nov, 56 ( Pt 11), 1501 - 4
The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidylyltransferase (RmlA), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa; Blankenfeldt W et al.; Glucose-1-phosphate thymidylyltransferase (RmlA; E.C . 2.7.7.24) is the first of four enzymes involved in the biosynthesis of dTDP-L-rhamnose, the precursor of L-rhamnose, a key component of the cell wall of many pathogenic bacteria . RmlA catalyses the condensation of thymidine triphosphate (dTTP) and alpha-D-glucose-1-phosphate (G1P), yielding dTDP-D-glucose . RmlA from Pseudomonas aeruginosa has been overexpressed and purified . Crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with PEG 6000 and lithium sulfate as precipitant . Several diffraction data sets of single frozen crystals were collected to a resolution of 1.66 A . Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 A, alpha = 89.9, beta = 80.9, gamma = 81.1 degrees . The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51% . Selenomethionine-labelled protein has been obtained and crystallized.

Int J Antimicrob Agents, 2000 Oct, 16(2), 131 - 3
Empirical treatment of sepsis in neutropenic patients; Klastersky J; Febrile neutropenia remains a major cause of morbidity in cancer patients receiving chemotherapy . Although the mortality associated with febrile neutropenia has dramatically decreased over the last three decades, the overall death rate during and immediately after an episode of febrile neutropenia can be as high as 10% with half of the patients dying directly as a result of the infection itself . A series of developments has led to this marked reduction in mortality . Among them, a pivotal role has been played by the concept of hospital-based empirical therapy with broad-spectrum combinations of antibiotics, aimed primarily against Gram-negative organisms, namely Pseudomonas aeruginosa

Int J Antimicrob Agents, 2000 Oct, 16(2), 103 - 6
Therapeutic guidelines for Pseudomonas aeruginosa infections; Giamarellou H; Pseudomonas aeruginosa nowadays is encountered among the leading pathogen in (i) ICU pneumonia; (ii) nosocomial bacteremia and AIDS primary bacteremia; (iii) iv drug users endocarditis; (iv) exacerbations of cystis fibrosis; (v) malignant external otitis and 'swimmers's ear', and (vi) contact lenses keratitis and traumatic endophthalmitis . The most vulnerable nosocomial hosts are the neutropenics and the mechanically ventilated patients in whom mortality rate exceeds 30% . Virulence of P . aeruginosa is attributed to the elaboration of various enzymes and toxins . There is also worldwide emergence of multiresistant phenotypes to antipseudomonal antibiotics . Therapeutic guidelines should therefore be based on (i) continuous resistance surveillance; (ii) in vitro synergistic interactions of antibacterial agents; (iii) pharmacodynamic properties of antibiotics interpreted by optimal dosing and appropriate frequency of administration; and (iv) current information on the necessity for combination therapy using an aminoglycoside.

J Bacteriol, 2000 Nov, 182(22), 6401 - 11
The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are controlled by quorum sensing and by RpoS; Winzer K et al.; In Pseudomonas aeruginosa, many exoproduct virulence determinants are regulated via a hierarchical quorum-sensing cascade involving the transcriptional regulators LasR and RhlR and their cognate activators, N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL) . In this paper, we demonstrate that the cytotoxic lectins PA-IL and PA-IIL are regulated via quorum sensing . Using immunoblot analysis, the production of both lectins was found to be directly dependent on the rhl locus while, in a lasR mutant, the onset of lectin synthesis was delayed but not abolished . The PA-IL structural gene, lecA, was cloned and sequenced . Transcript analysis indicated a monocistronic organization with a transcriptional start site 70 bp upstream of the lecA translational start codon . A lux box-type element together with RpoS (sigma(S)) consensus sequences was identified upstream of the putative promoter region . In Escherichia coli, expression of a lecA::lux reporter fusion was activated by RhlR/C4-HSL, but not by LasR/3O-C12-HSL, confirming direct regulation by RhlR/C4-HSL . Similarly, in P . aeruginosa PAO1, the expression of a chromosomal lecA::lux fusion was enhanced but not advanced by the addition of exogenous C4-HSL but not 3O-C12-HSL . Furthermore, mutation of rpoS abolished lectin synthesis in P . aeruginosa, demonstrating that both RpoS and RhlR/C4-HSL are required . Although the C4-HSL-dependent expression of the lecA::lux reporter in E . coli could be inhibited by the presence of 3O-C12-HSL, this did not occur in P . aeruginosa . This suggests that, in the homologous genetic background, 3O-C12-HSL does not function as a posttranslational regulator of the RhlR/C4-HSL-dependent activation of lecA expression.

Cytokine, 2000 Nov, 12(11), 1662 - 8
Role of cytokine-induced neutrophil chemoattractant-2 (CINC-2) alpha in a rat model of chronic bronchopulmonary infections with Pseudomonas aeruginosa; Amano H et al.; In order to investigate the role of the cytokine-induced neutrophil chemoattractant (CINC) in chronic bronchopulmonary infection, we developed a rat model of bronchopulmonary infection with Pseudomonas aeruginosa by using the agar bead method, and determined the kinetics of bacterial and cell number, as well as the concentrations of CINC-1, CINC-2, and CINC-3 in bronchoalveolar lavage (BAL) fluids in this model . The bacterial number in the lung rapidly increased from days 1 to 4, and declined 14 days after challenge . Neutrophil number in BAL fluid increased up to one day after challenge, and then slowly decreased during 14 days post-challenge . Among the CINCs, the local production of CINC-2 alpha sharply increased at day 1 and then decreased until day 4 post-challenge, while the local production of CINC-1 slightly increased at day 1 post-challenge . Neither CINC-2 beta nor CINC-3 were detected during the entire course of the infection . Increased CINC-2 mRNA expression in the lung tissue after challenge was associated with CINC-2 alpha production in BAL fluid . Moreover, an immunohistochemical study demonstrated the localization of CINC-1 and CINC-2 alpha primarily in alveolar macrophages and, to a much lesser extent, in bronchial epithelium of infected lung tissues, whereas CINC-2 beta and CINC-3 were not detected . When anti-CINC-1 or anti-CINC-2 alpha polyclonal antibodies were used for neutralizing neutrophil chemotactic activities in BAL fluids, the anti-CINC-2 alpha antibody inhibited 70% of the chemotactic activity in BAL fluids from infected rats at day 1 after challenge . No inhibition was observed by anti-CINC-1 antibody . These data indicate that CINC-2 alpha, which is produced by alveolar macrophages and bronchial epithelial cells, plays a pivotal role in neutrophil accumulation in the airway of a rat model of chronic bronchopulmonary infection with P . aeruginosa .

Braz J Med Biol Res, 2000 Nov, 33(11), 1275 - 82
Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity; Matsui T et al.; The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes . Expression of mycobacterial PLC-a and PLC-b in E . coli and M . smegmatis has been reported, whereas expression of the native proteins in M . tuberculosis H37Rv has not been demonstrated . The objective of the present study was to demonstrate that native PLC-a is expressed in M . tuberculosis H37Rv . Sera from mice immunized with recombinant PLC-a expressed in E . coli were used in immunoblots to evaluate PLC-a expression . The immune serum recognized a 49-kDa protein in immunoblots against M . tuberculosis extracts . No bands were visible in M . tuberculosis culture supernatants or extracts from M . avium, M . bovis and M . smegmatis . A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of beta-galactosidase activity . beta-Galactosidase activity was detected in M . smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages . The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive . In conclusion, expression of non-secreted native PLC-a was demonstrated in M . tuberculosis.

Clin Infect Dis, 2000 Oct, 31(4), E9 - E14 Epub 2000 Oct 25.
Large outbreak in a surgical intensive care unit of colonization or infection with Pseudomonas aeruginosa that overexpressed an active efflux pump; Bertrand X et al.; During a 30-month survey, 55 patients were colonized or infected by a single clone of Pseudomonas aeruginosa in a surgical intensive care unit (ICU) . This clone overexpressed an efflux pump system, and its antibiotic resistance pattern was extremely stable as it spread from patient to patient . Pulsed-field gel electrophoresis showed that isolates from different patients were genetically identical or very similar . We were unable to identify an environmental reservoir, but cultures of hand specimens from 2 health care workers were positive . It was not clear whether this carriage was the source of the epidemic or a consequence of it . However, the propagation of the epidemic clone was probably linked to its transmission by the staff from patient to patient . The outbreak was controlled, with difficulty, by strengthening isolation procedures, replacing the antiseptic soap being used by the staff, and changing the antibiotic prescription policy . This observation emphasizes the importance of compliance with hand washing and universal precautions.

J Microbiol Immunol Infect, 2000 Sep, 33(3), 154 - 8
Effects of quorum sensing signal molecules on the hydrogen peroxide resistance against planktonic Pseudomonas aeruginosa; Huang CT et al.; The effects of quorum sensing signal molecules in Pseudomonas aeruginosa, N-butanoyl-L-homoserinelactone (C4-HSL) and N-(3-oxododecanoyl)-L-homoserinelactone (3-oxo-C12-HSL) on planktonic cell resistance against hydrogen peroxide were studied . In P . aeruginosa JP2 cells with the deletion of lasI and rhlI, the viable cell concentration decreased with time and was reduced by about 4 log after 2 h of 7.5 mM H2O2 treatment, while only a 2-log reduction was found for the wild type P . aeruginosa PAO1 cells . When cultured with 20% PAO1 spent medium, P . aeruginosa JP2 showed similar hydrogen peroxide resistance to that seen in P aeruginosa PAO1 . Culturing with 20% JP2 spent medium or with 10 microM C4-HSL and 20 microM 3-oxo-C12-HSL did not affect P aeruginosa JP2 cell susceptibility to hydrogen peroxide . Although both 20% PAO1 and JP2 spent media reacted with H2O2 and reduced H2O2 to 50% of the strength of the original concentration, the remaining H2O2 was still sufficient to kill P . aeruginosa JP2 . These results indicate that the difference in cell resistance against H2O2 between P . aeruginosa PAO1 and JP2 was related to the existence of gene products of the lasI and rhlI systems . However, adding synthetic homoserine lactones alone did not increase P . aeruginosa JP2 cell resistance to H2O2 as seen in the experiments adding PAO1 spent medium . Determination of the detailed relation between cascade regulation in P . aeruginosa and its cell resistance to H2O2 will require further investigation.

Adv Perit Dial, 2000, 16, 195 - 7
Simultaneous removal and reinsertion of Tenckhoff catheters for the treatment of refractory exit-site infection; Lui SL et al.; Exit-site infection (ESI) refractory to medical therapy is an important complication of continuous ambulatory peritoneal dialysis (CAPD) . Between July 1994 and June 1998, 28 patients in our hospital underwent simultaneous removal and reinsertion of Tenckhoff catheters for the treatment of refractory ESI . The ESI was caused by Pseudomonas aeruginosa in 22 patients (78%), methicillin-resistant Staphylococcus aureus in 5 patients (18%), and diphtheroid bacilli in 1 patient (4%) . The patients had received antibiotic treatment for a mean duration of 11.6 +/- 5.8 weeks (range: 3-28 weeks) before their operations . During each operation, the old Tenckhoff catheter was removed and a new catheter was inserted in the opposite side of the abdomen . CAPD was resumed after two weeks of intermittent peritoneal dialysis . All patients received intravenous antibiotic cover for seven days after the operation . Early post-operative complications were uncommon . At one year after the operation, 22 patients (78%) were free of ESI . Six patients (22%) had a recurrence of ESI 21.3 +/- 6 weeks (range: 16-32 weeks) after their operation . All new infections were treated successfully with antibiotics . Seven patients (25%) had one episode of peritonitis each, all of which resolved with intraperitoneal antibiotic treatment . We conclude that simultaneous removal and reinsertion of Tenckhoff catheters is a safe and effective method for the treatment of refractory ESI . The procedure alleviates the need for temporary hemodialysis and allows an early return to CAPD.

J Microbiol Methods, 2000 Nov, 42(3), 245 - 53
Rapid detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes; Stender H et al.; A new chemiluminescent in situ hybridization (CISH) method that provides simultaneous detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water within 1 working day has been developed . Individual micro-colonies of P . aeruginosa were detected directly on membrane filters following 5 h of growth by use of soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeted to a species-specific sequence in P . aeruginosa rRNA . Within each micro-colony, reaction of the peroxidase with a chemiluminescent substrate generated light that was subsequently captured by film or with a digital camera system . Each spot of light represented one micro-colony of P . aeruginosa . Sensitivity and specificity for the identification of P . aeruginosa were 100% as determined by testing 28 P . aeruginosa strains and 17 other bacterial species that included closely related Pseudomonas species . Furthermore, the number of micro-colonies of P . aeruginosa represented by light spots correlated with counts of visible colonies following sustained growth . We conclude that PNA CISH speeds up traditional membrane filtration techniques and adds the specificity of PNA probe technology to generate fast and definitive results.

FEBS Lett, 2000 Oct 20, 483(2-3), 131 - 4
Antioxidative galloyl esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase; Abe I et al.; Gallic acid and its esters were evaluated as enzyme inhibitors of recombinant p-hydroxybenzoate hydroxylase (PHBH), a NADPH-dependent flavin monooxygenase from Pseudomonas aeruginosa . n-Dodecyl gallate (DG) (IC(50)=16 microM) and (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50)=16 microM), a major component of green tea polyphenols, showed the most potent inhibition, while product-like gallic acid did not inhibit the enzyme significantly (IC(50)>250 microM) . Inhibition kinetics revealed that both DG and EGCG inhibited PHBH in a non-competitive manner (K(I)=18.1 and 14.0 microM, respectively) . The enzyme inhibition was caused by specific binding of the antioxidative gallate to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction . Molecular modeling predicted that EGCG binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds.

Arch Microbiol, 2000 Sep, 174(3), 135 - 42
Pyoverdines: pigments, siderophores and potential taxonomic markers of fluorescent Pseudomonas species; Meyer JM; Pyoverdine, the yellow-green, water-soluble, fluorescent pigment of the fluorescent Pseudomonas species, is a powerful iron(III) scavenger and an efficient iron(III) transporter . As a fluorescent pigment, it represents a ready marker for bacterial differentiation and, as a siderophore, it plays an important physiological function in satisfying the absolute iron requirement of these strictly aerobic bacteria . Close to 40 structurally different pyoverdines have been identified to date, each characterized by a different peptidic part of the molecule and by a very narrow specificity as an iron transporter for Pseudomonas species, usually restricted to the producer strain or to strains producing an identical compound . Cross-reactivity does occur, however, for pyoverdines exhibiting partial identity at the peptide chain level, suggesting some information on the receptor-recognition site of the molecule . With the recent description of an operonic cluster of four genes involved in the synthesis of the chromophoric part of the molecule, a total of seven pyoverdine biosynthetic genes have been identified so far in Pseudomonas aeruginosa PAO1 . Although the precise function of the gene products needs further clarification, a biosynthetic pathway based on a multienzyme thiotemplate mechanism allowing a step-by-step synthesis of the whole chromopeptide molecule can be postulated . A promising future is expected from recent developments which indicate that pyoverdines might be considered as potent and easy-to-handle taxonomic markers for the fluorescent species of the genus Pseudomonas.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 107 - 12
Variation of the mexT gene, a regulator of the MexEF-oprN efflux pump expression in wild-type strains of Pseudomonas aeruginosa; Maseda H et al.; We found three variations of wild-type strains in terms of mexT-mediated regulation of the MexEF-OprN efflux pump, in which overexpression of the pump results in nfxC-type antibiotic resistance in Pseudomonas aeruginosa . Type-I: the mexT gene of the wild-type strain encoded inactive MexT and the nfxC-type mutants derived from this parent had an additional mutation in mexT converting MexT from the inactive to the active form . Type-II: The mexT gene in the wild-type strain had an 8-bp insert producing inactive MexT and the nfxC-type mutants from this parent had a deletion of the 8-bp insert converting inactive MexT to the active form . Type-III: Both the wild-type strain and its nfxC-type derivative produced identical and active MexT . The nfxC mutant from this parent must have an additional mutation . The original nfxC mutant isolated in 1990 might be derived from the Type-I parent strain.

Science, 2000 Oct 20, 290(5491), 527 - 30
CD95/CD95 ligand interactions on epithelial cells in host defense to Pseudomonas aeruginosa; Grassme H et al.; Pseudomonas aeruginosa causes severe infections, particularly of the lung, that are life threatening . Here, we show that P . aeruginosa infection induces apoptosis of lung epithelial cells by activation of the endogenous CD95/CD95 ligand system . Deficiency of CD95 or CD95 ligand on epithelial cells prevented apoptosis of lung epithelial cells in vivo as well as in vitro . The importance of CD95/CD95 ligand-mediated lung epithelial cell apoptosis was demonstrated by the rapid development of sepsis in CD95- or CD95 ligand-deficient mice, but not in normal mice, after P . aeruginosa infection.

Am J Vet Res, 2000 Oct, 61(10), 1204 - 8
Pharmacokinetics of ceftazidime in dogs following subcutaneous administration and continuous infusion and the association with in vitro susceptibility of Pseudomonas aeruginosa; Moore KW et al.; OBJECTIVE: To determine the pharmacokinetics of ceftazidime following subcutaneous administration and continuous IV infusion to healthy dogs and to determine the minimum inhibitory concentration (MIC) of ceftazidime for clinical isolates of Pseudomonas aeruginosa . ANIMALS: 10 healthy adult dogs . PROCEDURE: MIC of ceftazidime for 101 clinical isolates of P aeruginosa was determined in vitro . Serum concentrations of ceftazidime were determined following subcutaneous administration of ceftazidime (30 mg/kg of body weight) to 5 dogs and continuous IV infusion of ceftazidime (loading dose, 4.4 mg/kg; infusion rate, 4.1 mg/kg/h) for 36 hours to 5 dogs . RESULTS: The MIC of ceftazidime for P aeruginosa was < or = 8 microg/ml; all isolates were considered susceptible . Following SC administration of ceftazidime, mean beta disappearance half-life was 0.8 hours, and mean serum ceftazidime concentration exceeded the MIC for P aeruginosa for only 4.3 hours . Two dogs had gastrointestinal tract effects . Mean serum ceftazidime concentration exceeded 16 microg/ml during continuous IV infusion . None of the dogs developed adverse effects . CONCLUSIONS AND CLINICAL RELEVANCE: Administration of ceftazidime subcutaneously (30 mg/kg, q 4 h) or as a constant IV infusion (loading dose, 4.4 mg/kg; rate, 4.1 mg/kg/h) would maintain serum ceftazidime concentrations above the MIC determined for 101 clinical isolates of P aeruginosa . Use of these dosages may be appropriate for treatment of dogs with infections caused by P aeruginosa.

Antimicrob Agents Chemother, 2000 Nov, 44(11), 3003 - 7
Purification and biochemical characterization of the VIM-1 metallo-beta-lactamase; Franceschini N et al.; VIM-1 is a new group 3 metallo-beta-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area . In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla(VIM-1) gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step . The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing . Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor . VIM-1 hydrolyzes a broad array of beta-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-beta-lactamase inactivators . Only monobactams escape hydrolysis . The highest catalytic constant/K(m) ratios (>10(6) M(-1) . s(-1)) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem . Kinetic parameters showed remarkable variability with different beta-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these beta-lactam subfamilies . Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-beta-lactamases . Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.

Infect Immun, 2000 Nov, 68(11), 6202 - 8
Preparation and preclinical evaluation of a novel liposomal complete-core lipopolysaccharide vaccine; Bennett-Guerrero E et al.; Our objective is to develop a prophylactic vaccine strategy that can be evaluated for surgical and other high-risk hospitalized patients . In this paper, we describe the preparation and preclinical evaluation of a liposomal complete-core lipopolysaccharide (LPS) vaccine that is nontoxic and broadly antigenic . Complete-core (Ra-chemotype) LPSs were isolated from four gram-negative bacterial strains (Escherichia coli K-12, E . coli R1, Pseudomonas aeruginosa PAC608, and Bacteroides fragilis), mixed together to form a cocktail of complete-core LPSs, and then incorporated into multilamellar liposomes consisting of dimyristoyl phosphatidyl choline, dimyristoyl phosphatidylglycerol, and cholesterol in a 4:1:4 molar ratio . The endotoxic activities of these LPS-containing liposomes were less than 0.1% of the endotoxicities of the original free LPSs as measured by the Limulus amoebocyte lysate assay . In vivo administration of liposomal complete-core LPS mixed with Al(OH)(3) to rabbits resulted in no pyrogenicity or overt toxicity over a 7-day period . In immunoblots, sera from rabbits following active immunization elicited cross-reactive antibodies to a large panel of rough and smooth LPSs from numerous clinically relevant gram-negative bacteria, including E . coli (serotypes O1, O4, O6, O8, O12, O15, O18, O75, O86, O157, and O111), P . aeruginosa (Fisher-Devlin serotypes 1, 2, and 3, which correspond to International Antigenic Typing Scheme types 6, 11, and 2, respectively), Klebsiella pneumoniae (serotypes O1, O2ab, and O3), B . fragilis, and Bacteroides vulgatus . Active immunization of mice with liposomal complete-core LPS provided protection against a lethal challenge with E . coli O18 LPS . The vaccine tested was nontoxic, nonpyrogenic, and immunogenic against a wide variety of pathogens found in clinical settings.

Cochrane Database Syst Rev . 2000;(4):CD002009.
Once daily versus multiple daily dosing with intravenous aminoglycosides for cystic fibrosis; Tan K et al.; BACKGROUND: Patients with cystic fibrosis, who are chronically colonised with the organism Pseudomonas aeruginosa, often require repeated courses of intravenous aminoglycoside antibiotics for the management of pulmonary exacerbations . The properties of aminoglycosides suggest that they could be given in higher concentrations less often . OBJECTIVES: To assess the effectiveness and safety of once-daily versus multiple-daily dosing of intravenous aminoglycoside antibiotics for the management of pulmonary exacerbations in cystic fibrosis . SEARCH STRATEGY: We searched the Cystic Fibrosis specialist trials register held at the Cochrane Cystic Fibrosis and Genetic Disorders Group's editorial base, which comprises references identified from comprehensive electronic database searches, handsearching relevant journals and handsearching abstract books of conference proceedings . Date of the most recent search: February 2000 . SELECTION CRITERIA: All randomised controlled trials, whether published or unpublished, in which once-daily dosing of aminoglycosides has been compared with multiple-daily dosing in terms of efficacy and/or toxicity, in patients with cystic fibrosis . DATA COLLECTION AND ANALYSIS: Both reviewers independently extracted data and assessed trial quality . Authors of one study were contacted to obtain missing information . MAIN RESULTS: Two trials reporting results from a total of 70 patients were included in this review . Both trials compared once-daily dosing with thrice-daily dosing reporting data on Forced Expiratory Volume at one second (FEV1), Forced Vital Capacity (FVC), nutritional status and side effects . There was no significant difference in efficacy or in the incidence of ototoxicity and nephrotoxicity between treatment groups . REVIEWER'S CONCLUSIONS: Despite a lack of difference between the two groups we feel that these results should be viewed with caution as the numbers of patients involved was small and lacks the power to detect a difference between the groups . This systematic review has highlighted the need for a well designed, adequately-powered, multicentre, randomised controlled trial assessing the efficacy of once-daily versus multiple-daily dosing of intravenous aminoglycosides for pulmonary exacerbations in cystic fibrosis.

Cochrane Database Syst Rev . 2000;(4):CD001917.
Home intravenous antibiotics for cystic fibrosis; Marco T et al.; BACKGROUND: Recurrent endobronchial infection in cystic fibrosis requires treatment with intravenous antibiotics for several weeks, which is usually administered in hospital, affecting health costs and quality of life for patients and their families . It is not known whether patients receiving intravenous treatment at home have better or equivalent health outcomes, if costs are reduced or if it is preferred than in-hospital treatment . Home treatment requires training to patients and carers and usually needs a few previous days in hospital . OBJECTIVES: To determine whether home intravenous antibiotic therapy in cystic fibrosis is as effective as in-patient intravenous antibiotic therapy and if it is preferred by patients and/or families . SEARCH STRATEGY: References to trials were obtained from the specialist cystic fibrosis trials register held by the editorial base of the Cochrane Cystic Fibrosis and Genetic Disorders Group . Handsearching of the abstracts books of all Spanish Conferences on cystic fibrosis and the last European Conference (Stockholm, 2000) was carried out by authors . SELECTION CRITERIA: Randomised controlled trials where home intravenous antibiotic treatment for patients with cystic fibrosis was compared with in-hospital intravenous antibiotic treatment, including adults and children with cystic fibrosis . All kinds of antibiotics and regimens administered intravenous were included . DATA COLLECTION AND ANALYSIS: Three reviewers independently selected the trials to be included in the review, assessed methodological quality of each trial and extracted data using a standardised form . Because of several limitations, narrative synthesis was used at this stage . MAIN RESULTS: One study was included with 17 patients aged 10 to 41 years with an infective exacerbation by Pseudomonas aeruginosa . All their 31 admissions were analysed as independent events . Outcomes were measured at 21 days of follow-up after initiation of treatment . Home patients had fewer investigations performed than hospital patients (p<0.002) and general activity was higher in the home group . No differences were found for clinical outcomes, adverse events, complications of intravenous lines or line changes or time to next admission . Home patients received less low-dose home maintenance antibiotic . Quality of life measures showed no differences for dyspnoea and emotional state, but fatigue and mastery were worse for home patients, possibly due to a higher general activity and need of support . Personal, family, sleeping and eating disruptions were less important for home than hospital admissions . Home therapy was cheaper for families and the hospital . Indirect costs were not determined . REVIEWER'S CONCLUSIONS: The current evidence is restricted to one small study . It suggests that in the short term home therapy does not harm patients and in general reduces social disruptions . The decision to attempt home treatment should be based on an individual basis and appropriate local resources . More research is urgently required.

J Immunol, 2000 Oct 1, 165(7), 3941 - 50
Role of cystic fibrosis transmembrane conductance regulator in pulmonary clearance of Pseudomonas aeruginosa in vivo; Chroneos ZC et al.; Cystic fibrosis (CF)2 is a fatal genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) that is commonly associated with chronic pulmonary infections with mucoid Pseudomonas aeruginosa (PA) . To test the hypothesis that CFTR plays a direct role in PA adhesion and clearance, we have used mouse lines expressing varying levels of human (h) or mouse (m) CFTR . A subacute intratracheal dose of 3 x 10(6) bacteria was cleared with similar kinetics in control wild-type (WT) and transgenic mice overexpressing hCFTR in the lung from the surfactant protein C (SP-C) promoter (SP-C-hCFTR+/-) . In a second series of experiments, the clearance of an acute intratracheal dose of 1.5 x 10(7) PA bacteria was also similar in WT, hemizygous SP-C-hCFTR+/-, and bitransgenic gut-corrected FABP-hCFTR+/+-mCFTR-/-, the latter lacking expression of mCFTR in the lung . However, a small but significant decrease in bacterial killing was observed in lungs of homozygote SP-C-hCFTR+/+ mice . Lung pathology in both WT and SP-C-hCFTR+/+ mice was marked by neutrophilic inflammation and bacterial invasion of perivascular and subepithelial compartments . Bacteria were associated primarily with leukocytes and were not associated with alveolar type II or bronchiolar epithelial cells, the cellular sites of SP-C-hCFTR+/+ transgene expression . The results indicate that there is no direct correlation between levels of CFTR expression and bacterial clearance or association of bacteria with epithelial cells in vivo.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 329 - 33
Tools for discovery of inhibitors of the 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase: an approach with enzymes from the pathogenic bacterium Pseudomonas aeruginosa; Altincicek B et al.; Two Pseudomonas aeruginosa genes encoding the enzymes 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase, both involved in the mevalonate-independent biosynthesis of isoprenoids, have been expressed as recombinant enzymes in Escherichia coli . The purified P . aeruginosa DXP reductoisomerase was inhibited by submicromolar concentrations of the antibiotics fosmidomycin and FR-900098 in a well established method . A novel and convenient spectrophotometric assay was developed to determine activity and inhibition of P . aeruginosa DXP synthase . Fluoropyruvate is described as a first inhibitor of DXP synthase.

Jpn J Ophthalmol, 2000 Sep-Oct, 44(5), 494 - 502
Role of Pseudomonas aeruginosa culture filtrates in the association, invasion, and cytotoxicity against cloned cells from murine corneal epithelium and KB cells; Ishino G et al.; PURPOSE: To clarify the effect of Pseudomonas aeruginosa culture filtrates on the association with, invasion into, and cytotoxicity against cloned cells from murine corneal epithelial cells and KB cells . METHODS: Simian virus 40-transformed murine corneal epithelial (MCE) cells were established . Murine corneal epithelial cells and KB cells were infected with a protease-positive strain, IID1117 (Pa IID1117), and a protease-negative strain, IID1130 (Pa IID1130) of P . aeruginosa, and then tested for association and invasion of Pa IID1117 . The cytotoxicity test was performed by incubating the cells with culture filtrate . RESULTS: Association of Pa IID1117 with KB cells pretreated with Pa IID1130 was significantly promoted . After pretreatment with culture filtrate, invasion was more effective into MCE cells than into KB cells . When infecting bacteria (Pa IID1117) were pretreated with protease inhibitor, invasion of the bacteria into MCE cells and KB cells clearly decreased . The cellular damage induced by the culture filtrate of Pa IID1130 was greater than the damage by that of Pa IID1117 . CONCLUSION: These results suggest that association of P . aeruginosa with MCE cells and KB cells was influenced by the culture filtrates other than proteases, and that invasion of P . aeruginosa into MCE cells and KB cells was promoted by protease.

Pharmazie, 2000 Sep, 55(9), 651 - 8
{Synthesis and transformations of ethyl 1,4-dihydro-4-oxo(1)benzofuro(3,2-b)pyridine-3-carboxylic acid esters: new antibacterial agents}; Gorlitzer K et al.; The title compound 7 was synthesized from potassium 3-amino-{1}benzofuran-2-carboxylate (1) by Gould-Jacobs-reaction . The pyridone 7 reacted with ethyl iodide by N- and O-alkylation to give 9 and 10, while methyl iodide only yielded the N-methylpyridone 11 . The 4-chloropyridine 15 was obtained by heating 7 in phosphoryl chloride . Alkaline saponification of the esters 7, 9-11 and 15 afforded the carboxylic acids 8, 12-14 and 16 . The carbaldehydes 19 and 22 were prepared from the ethylesters 10 and 15 by boranate reduction to the carbinoles 17 and 20 followed by dehydrogenation with activated manganese dioxide . The aldehyde 22 reacted with beta-aminocrotonic acid esters to yield the 1,4 dihydropyridines (DHP) 23 . The pyridines 24 were formed by chemical or electrochemical dehydrogenation of the DHP 23 . The tetrazole 27 was accessible from the aldehyde 22 via the aldoxime 25 and the nitrile 26 . The pyridone 7 reacted with tosylisocyanate to yield the 4-tosylaminopyridine 28, which after alkylation to form 29 followed by detosylation to give 30 and subsequent alkaline hydrolysis produced the 4-aminonicotinic acid 31 . The investigation The N-ethylpyridone-3-carboxylic acid 12 showed antibacterial activity comparable to the reference substance nalidixic acid against Pseudomonas aeruginosa, Escherichia coli and Bacillus megaterium.

J Bacteriol, 2000 Nov, 182(21), 6066 - 74
Sequence of the genome of the temperate, serotype-converting, Pseudomonas aeruginosa bacteriophage D3; Kropinski AM; Temperate bacteriophage D3, a member of the virus family Siphoviridae, is responsible for serotype conversion in its host, Pseudomonas aeruginosa . The complete sequence of the double-stranded DNA genome has been determined . The 56,426 bp contains 90 putative open reading frames (ORFs) and four genes specifying tRNAs . The latter are specific for methionine (AUG), glycine (GGA), asparagine (AAC), and threonine (ACA) . The tRNAs may function in the translation of certain highly expressed proteins from this relatively AT-rich genome . D3 proteins which exhibited a high degree of sequence similarity to previously characterized phage proteins included the portal, major head, tail, and tail tape measure proteins, endolysin, integrase, helicase, and NinG . The layout of genes was reminiscent of lambdoid phages, with the exception of the placement of the endolysin gene, which parenthetically also lacked a cognate holin . The greatest sequence similarity was found in the morphogenesis genes to coliphages HK022 and HK97 . Among the ORFs was discovered the gene encoding the fucosamine O-acetylase, which is in part responsible for the serotype conversion events.

J Bacteriol, 2000 Nov, 182(21), 5990 - 6
Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili; Kohler T et al.; We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities . Swarming in P . aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids . Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming . Cells from the edge of the swarm were about twice as long as cells from the swarm center . In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy . While a fliC mutant of P . aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm . Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm . Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P . aeruginosa in this type of surface motility.

Am J Infect Control, 2000 Oct, 28(5), 347 - 51
Microbiologic contamination study of nebulizers after aerosol therapy in patients with cystic fibrosis; Vassal S et al.; BACKGROUND: To evaluate the contamination of delivery systems after an aerosol therapy session in patients with cystic fibrosis who have chronic Pseudomonas aeruginosa infection . METHODS: Fifty-three patients with cystic fibrosis were enrolled in the study from March 1996 to June 1997 . All patients were age 7 years or older and had P aeruginosa infection . They also had been treated with recombinant deoxyribonuclease and were capable of producing sputum for culture . RESULTS: Nine devices were excluded for the study . A total of 44 nebulizers were included: 37 from patients with P aeruginosa colonization with a count of 10(6) colony-forming units/mL or more and 7 with a count of between 10(5) colony-forming units/mL and 10(6) colony-forming units/mL . CONCLUSION: This study demonstrates that in the absence of cleaning, nebulizers of patients with cystic fibrosis who are infected with P aeruginosa are likely to be contaminated by a pathogenic flora.

Am J Infect Control, 2000 Oct, 28(5), 333 - 9
Ventilator-associated pneumonia in very low-birth-weight infants at the time of nosocomial bloodstream infection and during airway colonization with Pseudomonas aeruginosa; Cordero L et al.; PURPOSE: To study retrospectively the incidence of ventilator-associated pneumonia (VAP) at the time of Pseudomonas aeruginosa nosocomial bloodstream infection (BSI) and at the time of P aeruginosa airway colonization.Materials and Methods: Fifteen very low-birth-weight infants who had P aeruginosa BSI and 33 others who did not but who had P aeruginosa airway-colonization were studied . We correlated clinical data, blood cultures (BCs), and tracheal cultures (TCs) with radiologic findings from radio-graphs taken within 2 days before, the day of, and 1 day after BCs or TCs were first positive for P aeruginosa . Chest radiographs were graded by using semiquantitative scores for bronchopulmonary dysplasia and for pneumonia . RESULTS: Mean birth weight, gestational age, and age when BC or TC became positive were similar for patients with BSI and colonization . At the time of BSI, 2 infants had airway colonization with P aeruginosa; the TCs of the remaining 13 grew P aeruginosa as a new pathogen . Thirteen of 15 patients with BSI, but none of 33 infants with colonization, died within 2 days of positive BC . VAP was diagnosed in 13 of 15 patients with BSI and in 3 of 33 infants with colonization . CONCLUSION: Mechanically ventilated very low-birth-weight infants whose TCs yield P aeruginosa but whose BCs remain negative infrequently have VAP are presumed airway-colonized and are expected to survive . Conversely, VAP is likely to be found when BCs and TCs simultaneously grow P aeruginosa, and high mortality is anticipated.

J Mol Evol, 2000 Sep, 51(3), 205 - 13
A new appraisal of the prokaryotic origin of eukaryotic phytochromes; Herdman M et al.; The evolutionary origin of the phytochromes of eukaryotes is controversial . Three cyanobacterial proteins have been described as "phytochrome-like" and have been suggested to be potential ancestors of these essential photoreceptors: Cph1 from Synechocystis PCC 6803, showing homology to phytochromes along its entire length and known to attach a chromophore; and PlpA from Synechocystis PCC 6803 and RcaE from Fremyella diplosiphon, both showing homology to phytochromes most strongly only in the C-terminal region and not known to bind a chromophore . We have reexamined the evolution of the photoreceptors using for PCR amplification a highly conserved region encoding the chromophore-binding domain in both Cph1 and phytochromes of plants and have identified genes for phytochrome-like proteins (PLP) in 11 very diverse cyanobacteria . The predicted gene products contain either a Cys, Arg, Ile, or Leu residue at the putative chromophore binding site . In 10 of the strains examined only a single gene was found, but in Calothrix PCC 7601 two genes (cphA and cphB) were identified . Phylogenetic analysis revealed that genes encoding PLP are homologues that share a common ancestor with the phytochromes of eukaryotes and diverged before the latter . In contrast, the putative sensory/regulatory proteins, including PlpA and RcaE, that lack a part of the chromophore lyase domain essential for chromophore attachment on the apophytochrome, are only distantly related to phytochromes . The Ppr protein of the anoxygenic photosynthetic bacterium Rhodospirillum centenum and the bacterial phytochrome-like proteins (BphP) of Deinococcus radiodurans and Pseudomonas aeruginosa fall within the cluster of cyanobacterial phytochromes.

Diagn Microbiol Infect Dis, 2000 Sep, 38(1), 37 - 41
Activity of piperacillin/tazobactam in combination with amikacin, ciprofloxacin, and trovafloxacin against Pseudomonas aeruginosa by time-kill; Burgess DS et al.; Pseudomonal infections have a high rate of morbidity and mortality, thus combination therapy is often recommended . We compared the activity of piperacillin/tazobactam in combination with amikacin, ciprofloxacin, or trovafloxacin at different concentrations against P . aeruginosa using time-kill methodology . MICs were determined for 4 clinical isolates of P . aeruginosa . Time-kill studies were conducted over 24 h . Each drug was tested alone and in combination using the following concentrations: 2 and 1/4, 1/4 and 2, and 1/4 and 1/4xMIC of piperacillin/tazobactam and amikacin, ciprofloxacin, or trovafloxacin . Combinations were classified as synergistic, indifferent, or antagonistic . Synergy was defined as > or = 2-log(10) decrease in CFU/mL at 24 h with the combination when compared to the most active single agent and the number of surviving organisms for the antimicrobial combination was > or =2-log(10) less than the initial inoculum . The MICs for piperacillin/tazobactam, amikacin, ciprofloxacin, and trovafloxacin, ranged from 4/4-512/4, 0.5-4, 0.125-4, and 0.5-8 microg/mL, respectively . Fifty eight percent of the combinations using concentrations of 1/4xMIC of piperacillin/tazobactam and 2xMIC of amikacin, ciprofloxacin, and trovafloxacin or 2xMIC of piperacillin/tazobactam and 1/4xMIC of amikacin, ciprofloxacin, and trovafloxacin were synergistic . Although no differences existed in synergistic activity between the two combinations, the 1/4 and 2xMIC maintained colony counts below the limit of quantification for 24 h for a significantly greater percentage of isolates than the 2 and 1/4xMIC combinations (75 and 25%, respectively; p = 0.04) . Overall, synergy was most frequently (42%) noted with the piperacillin/tazobactam and amikacin combinations followed by 33 and 8% of the piperacillin/tazobactam and trovafloxacin and ciprofloxacin combinations . No combination demonstrated antagonism . Further more extensive studies are necessary to determine clinical significance.

Burns, 2000 Dec, 26(8), 737 - 40
Nosocomial infections in an Iranian burn care center; Lari AR et al.; Burn patients are obviously at high risk for nosocomial infections due to the immunocompromizing effects of burn injury . Pseudomonas aeruginosa is an important life-threatening nosocomial pathogen in burn units . The aim of this study was to determine nosocomial infections in the Tohid Burn Center in Tehran, Iran . Materials of this study were samples of burn wounds and blood from 582 patients who required hospitalization during March 1996 and September 1998 . Burn wound samples were taken on admission day, 3 and 7 days after admission . Frequency of culture positive on admission day, 3 and 7 days after admission were 15, 66, and 88%, respectively . Frequency of P . aeruginosa and Staphylococcus aureus on admission day were 35 and 34%, on the third day after admission 73 and 15%, and at the end of the first week of admission 87 and 9%, respectively . Frequency of blood culture positive was 36% (19/53) of which 89% were P . aeruginosa . Overall mortality rate was 18.5% (108/582) . Of these patients, frequency of positive wound culture was 92% (99/108) . In conclusion, our results show that P . aeruginosa is the leading cause of nosocomial infections in our burn center . It is also necessary to introduce urgent measures for restriction of the spread of P . aeruginosa infections in our burn center.

J Hosp Infect, 2000 Sep, 46(1), 23 - 30
An outbreak of multidrug-resistant Pseudomonas aeruginosa infection associated with contamination of bronchoscopes and an endoscope washer-disinfector; Schelenz S et al.; Over a two-month period, two distinct types of Pseudomonas aeruginosa resistant to ceftazidime and azlocillin were isolated from bronchial specimens of ITU patients who had been previously bronchoscoped . The source of the outbreak was probably a faulty contaminated bronchoscope washer-disinfector which had been purchased a year earlier but not properly maintained . This paper describes the outbreak, the identification and elimination of the source, and the steps taken to prevent recurrence . Several automated, closed washer-disinfectors had been bought by the hospital in response to health and safety concerns about glutaraldehyde disinfection toxicity, but the operation and maintenance of these machines had not been supervised . Several other washer-disinfectors were also found to be faulty . The potential hazards of automated endoscope washer-disinfectors and the importance of controlled professional maintenance, servicing and training is discussed.

Microbiology, 2000 Oct, 146 ( Pt 10), 2543 - 54
Role of Pseudomonas aeruginosa PhoP-phoQ in resistance to antimicrobial cationic peptides and aminoglycosides; Macfarlane EL et al.; Resistance to the polycationic antibiotic polymyxin B and expression of the outer-membrane protein OprH in the opportunistic pathogen Pseudomonas aeruginosa both involve the PhoP-PhoQ two-component regulatory system . The genes for this system form an operon with oprH, oprH-phoP-phoQ, that responds to Mg(2+) starvation and PhoP levels . In this study, the Mg(2+)-regulated promoter for this operon was mapped upstream of oprH by primer-extension experiments . An oprH::xylE-Gm(R) mutant H855 was constructed and measurement of the catechol 2,3-dioxygenase activity expressed from this transcriptional fusion provided evidence for a second, weak promoter for phoP-phoQ . Wild-type P . aeruginosa PAO1 strain H103 was found to exhibit Mg(2+)-regulated resistance to the alpha-helical antimicrobial cationic peptide CP28 in addition to its previously characterized resistance to polymyxin B . Resistance to this peptide was unchanged in the OprH-null mutant H855 and a PhoP-null mutant H851 . In contrast, PhoQ-null mutant H854 demonstrated constitutive CP28 resistance . Northern blot analysis revealed constitutive expression of phoP in this strain, implicating PhoP-PhoQ in the resistance of P . aeruginosa to cationic peptides . Furthermore, all three null-mutant strains demonstrated increased resistance to the aminoglycoside antibiotics streptomycin, kanamycin and amikacin . Two additional mutant strains, H895 and H896, were constructed that carried unmarked deletions in oprH and were found to exhibit aminoglycoside susceptibility equivalent to that of the wild-type . This result provided definitive evidence that OprH is not involved in P . aeruginosa aminoglycoside resistance and that the changes in resistance in strain H855 and a previously reported oprH mutant were due to polar effects on phoP-phoQ rather than loss of OprH expression . A role for PhoP-PhoQ in resistance to aminoglycosides is envisaged that is distinct from that in resistance to cationic peptides and polymyxin B.

Microbiology, 2000 Oct, 146 ( Pt 10), 2531 - 41
Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of exoS; Kaufman MR et al.; Pseudomonas aeruginosa is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis . Using a tissue culture system, invasive strains of P . aeruginosa were discovered to induce apoptosis at high frequency in HeLa and other epithelial and fibroblast cell lines . This apoptotic phenotype in the infected cells was determined by several criteria including (i) visual changes in cell morphology, (ii) induction of chromatin condensation and nuclear marginalization, (iii) the presence of a high percentage of cells with subG1 DNA content, and (iv) activation of caspase-3 activity . Induction of the type III secretion machinery, but not invasion of P . aeruginosa is required for induction of apoptosis . The apoptosis phenotype is independent of the cytoskeletal rearrangements that occur in the host cell early after infection . Mutants in P . aeruginosa exoS fail to induce apoptosis and complementation with wild-type exoS restored the apoptosis-inducing capacity, demonstrating that ExoS is the effector molecule . Analysis of exoS activity mutants shows that the ADP-ribosylating capacity of ExoS is essential for inducing the apoptotic pathway.

Microbiology, 2000 Oct, 146 ( Pt 10), 2521 - 30
Secreted products of a nonmucoid Pseudomonas aeruginosa strain induce two modes of macrophage killing: external-ATP-dependent, P2Z-receptor-mediated necrosis and ATP-independent, caspase-mediated apoptosis; Zaborina O et al.; A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium . These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium . Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5'-nucleotidase (ATPase and/or phosphatase), were detected in the medium . These ATP-utilizing enzymes are believed to convert external ATP, presumably effluxed from macrophages, to various adenine nucleotides, which then activate purinergic receptors such as P2Z, leading to enhanced macrophage cell death . Pretreatment of macrophages with periodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z receptor activation, prevented subsequent ATP-induced macrophage cell death . A second type of cytotoxic factor(s) operated in an ATP-independent manner such that it triggered activation of apoptotic processes in macrophages, leading to proteolytic conversion of procaspase-3 to active caspase-3 . This cytotoxic factor(s) did not appear to act on procaspase-3 present in macrophage cytosolic extracts . Intact macrophages, when exposed to the cytotoxic factor(s) for 6-16 h, underwent apoptosis and demonstrated the presence of active caspase-3 in their cytosolic extracts . Interestingly, two redox proteins, azurin and cytochrome c(551), were detected in the cytotoxic preparation . When cell-line-derived or peritoneal macrophages or mast cells were incubated overnight with Q-Sepharose column flow-through fraction or with a mixture of azurin and cytochrome c(551), they underwent extensive cell death due to induction of apoptosis.

Microbiology, 2000 Oct, 146 ( Pt 10), 2509 - 19
migA, a quorum-responsive gene of Pseudomonas aeruginosa, is highly expressed in the cystic fibrosis lung environment and modifies low-molecular-mass lipopolysaccharide; Yang H et al.; Pseudomonas aeruginosa is an opportunistic human pathogen which poses a major threat to patients with cystic fibrosis (CF) . Excessive amounts of mucus present in the lungs of CF patients promotes the colonization of P . aeruginosa . The migA gene, encoding a putative glycosyltransferase, has been shown to be highly inducible by respiratory mucus derived from CF patients . In this study, it is further demonstrated by population transcript analysis that the migA gene is highly expressed in the CF lung environment . Deletion analysis of the migA promoter identified a las-box-like sequence commonly found in promoters that are responsive to quorum sensing regulation . Further analysis of migA expression in quorum-sensing-defective strains, as well as its expression in response to autoinducer molecules, demonstrated that migA is regulated by the RhlI/RhlR quorum sensing regulatory system . Functionally, as the MigA sequence homology data suggested, the migA gene indeed affects the structure of LPS in P . aeruginosa . Increased expression of the migA gene results in a loss of core-plus-one LPS, while having no obvious effect on the long-chain O-antigen-bearing LPS . Although the exact biological role of the core-plus-one LPS is not clear, these experimental results suggest that migA up-regulation in the CF lung environment is part of the adaptive response which confers on P . aeruginosa a survival advantage.

Microbiology, 2000 Oct, 146 ( Pt 10), 2495 - 508
Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry; Hanna SL et al.; Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates . To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography-tandem mass spectrometry (microLC/MS/MS) . The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections . Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis . Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels . Fourteen proteins were selected for analysis; this group included those whose expression was common between both strains as well as unique for each strain . The proteins were identified by microLC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192.

Microbiology, 2000 Oct, 146 ( Pt 10), 2481 - 93
Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa; Wu H et al.; The pathogenesis of Pseudomonas aeruginosa is associated with expression of virulence factors, many of which are controlled by two N:-acylhomoserine lactone (AHL)-based quorum-sensing systems . Escherichia coli strains equipped with a luxR-based monitor system expressing green fluorescent protein (GFP) in the presence of exogenous AHL molecules were used to detect the production of AHLs from P . aeruginosa in vivo . Mice were challenged intratracheally with alginate beads containing P . aeruginosa and E . coli and killed on different days after the challenge . By means of confocal scanning laser microscopy, GFP-expressing E . coli bacteria could be detected in the lung tissues, indicating production and excretion of AHL molecules in vivo by the infecting P . aeruginosa . AHL signals were detected mainly in lung tissues exhibiting severe pathological changes . These findings support the view that expression of AHL molecules by P . aeruginosa during infection coincides with its pathogenesis.

Microbiology, 2000 Oct, 146 ( Pt 10), 2425 - 34
Vanadium interferes with siderophore-mediated iron uptake in Pseudomonas aeruginosa; Baysse C et al.; Vanadium is a metal that under physiological conditions can exist in two oxidation states, V(IV) (vanadyl ion) and V(V) (vanadate ion) . Here, it was demonstrated that both ions can form complexes with siderophores . Pseudomonas aeruginosa produces two siderophores under iron-limiting conditions, pyoverdine (PVD) and pyochelin (PCH) . Vanadyl sulfate, at a concentration of 1-2 mM, strongly inhibited growth of P . aeruginosa PAO1, especially under conditions of severe iron limitation imposed by the presence of non-utilizable Fe(III) chelators . PVD-deficient mutants were more sensitive to vanadium than the wild-type, but addition of PVD did not stimulate their growth . Conversely, PCH-negative mutants were more resistant to vanadium than the wild-type strain . Both siderophores could bind and form complexes with vanadium after incubation with vanadyl sulfate (1:1, in the case of PVD; 2:1, in the case of PCH) . Although only one complex with PVD, V(IV)-PVD, was found, both V(IV)- and V(V)-PCH were detected . V-PCH, but not V-PVD, caused strong growth reduction, resulting in a prolonged lag phase . Exposure of PAO1 cells to vanadium induced resistance to the superoxide-generating compound paraquat, and conversely, exposure to paraquat increased resistance to V(IV) . Superoxide dismutase (SOD) activity of cells grown in the presence of V(IV) was augmented by a factor of two . Mutants deficient in the production of Fe-SOD (SodB) were particularly sensitive to vanadium, whilst sodA mutants deficient for Mn-SOD were only marginally affected . In conclusion, it is suggested that V-PCH catalyses a Fenton-type reaction whereby the toxic superoxide anion O(2)- is generated, and that vanadium compromises PVD utilization.

Microbiology, 2000 Oct, 146 ( Pt 10), 2365 - 73
Monitoring genome evolution ex vivo: reversible chromosomal integration of a 106 kb plasmid at two tRNA(Lys) gene loci in sequential Pseudomonas aeruginosa airway isolates; Kiewitz C et al.; The genome rearrangements in sequential Pseudomonas aeruginosa clone K isolates from the airways of a patient with cystic fibrosis were determined by an integrated approach of mapping, sequencing and bioinformatics . Restriction mapping uncovered an 8.9 kb deletion of PAO sequence between phnAB and oprL in clone K, and two 106 kb insertions either adjacent to this deletion or several hundred kilobases away, close to the pilA locus . These 106 kb blocks of extra DNA also co-existed as the circular plasmid pKLK106 in several clone K isolates and were found to be closely related to plasmid pKLC102 in P . aeruginosa clone C isolates . The breakpoints of the deletion in clone K and the attB-attP sequences for the reversible integration of the plasmid in clones C and K were located within the 3' end of the lysine tRNA structural genes (att site) . pKLK106 sequentially recombined with either of the two tRNA(Lys) genes in clone K isolates . The att site of the pilA hypervariable region has been utilized by clone C to target its plasmid pKLC102 into the chromosome; the att site of the phnAB-oprL region has been employed by strain PAO to incorporate a DNA block encoding pyocin, transposases and IS elements . The use of typical phage attachment sites by conjugative genetic elements could be one of the major mechanisms used by P . aeruginosa to generate the mosaic genome structure of blocks of species-, clone- and strain-specific DNA . The example described here demonstrates the potential impact of systematic genome analysis of sequential isolates from the same habitat on our understanding of the evolution of microbial genomes.

Microbiology, 2000 Oct, 146 ( Pt 10), 2351 - 64
An interactive web-based Pseudomonas aeruginosa genome database: discovery of new genes, pathways and structures; Croft L et al.; Using the complete genome sequence of Pseudomonas: aeruginosa PAO1, sequenced by the Pseudomonas: Genome Project (ftp://ftp.pseudomonas . com/data/pacontigs.121599), a genome database has been developed containing information on more than 95% of all ORFs in Pseudomonas: aeruginosa . The database is searchable by a variety of means, including gene name, position, keyword, sequence similarity and Pfam domain . Automated and manual annotation, nucleotide and peptide sequences, Pfam and SMART domains (where available), Medline and GenBank links and a scrollable, graphical representation of the surrounding genomic landscape are available for each ORF . Using the database has revealed, among other things, that P . aeruginosa contains four chemotaxis systems, two novel general secretion pathways, at least three loci encoding F17-like thin fimbriae, six novel filamentous haemagglutinin-like genes, a number of unusual composite genetic loci related to vgr/RHS: elements in Escherichia coli, a number of fix-like genes encoding a micro-oxic respiration system, novel biosynthetic pathways and 38 genes containing domains of unknown function (DUF1/DUF2) . It is anticipated that this database will be a useful bioinformatic tool for the Pseudomonas: community that will continue to evolve.

Microbiol Immunol, 2000, 44(8), 629 - 35
Antibacterial properties of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide-specific monoclonal antibody (MAb) in a murine thigh infection model: combined effects of MAb and ceftazidime; Akiyama M et al.; A murine monoclonal antibody (MAb) specific for the Pseudomonas aeruginosa immunotype 1 (It-1) lipopolysaccharide (LPS) O-side chain was evaluated in terms of its in vitro bactericidal opsonophagocytic activity and in vivo bacterial killing in a mouse thigh infection model . An immunoglobulin (Ig) G2a MAb Ld3-2F2, specific for It-1 LPS, mediated in vitro complement-dependent opsonophagocytic killing at a concentration of 10 microg/ml . MAb-mediated, complement-dependent killing also occurred in the absence of neutrophils at serum concentrations in excess of 20% . A remarkable synergy was observed in opsonophagocytic assays between MAb Ld3-2F2 (0.5 microg/ml) and ceftazidime (1/4 MIC) . The administration of MAb Ld3-2F2 at a level of 1 microg resulted in a significant decrease in the number of bacteria in the thigh muscles of normal mice, while 100 microg of the same MAb was required for one log of reduction in the number of bacteria at the same site in neutropenic mice . The combined therapy with MAb Ld3-2F2 and ceftazidime provided a significant reduction in the density of bacteria in the thigh muscle at 9 hr post-infection in normal and neutropenic mice as compared with those after treatment alone or with no treatment (P< 0.01) . These favorable in vitro and in vivo interactions of an LPS-specific IgG MAb and ceftazidime strongly support their potential for use in therapy, combined with an LPS-reactive MAb and parenteral antipseudomonas beta-lactam antibiotics in the therapy of systemic Pseudomonas infections in normal and neutropenic hosts.

Ann Emerg Med, 2000 Oct, 36(4), 383 - 7
Ecthyma gangrenosum as a manifestation of Pseudomonas sepsis in a previously healthy child; Mull CC et al.; We present a case of Pseudomonas aeruginosa sepsis heralded by ecthyma gangrenosum in a previously healthy 15-month-old child . Pseudomonas infection and its uncommon skin manifestation are rarely encountered in an immunocompetent child . This case highlights the critical importance of identifying ecthyma gangrenosum to institute optimal antimicrobial therapy.

Jpn J Antibiot, 2000 Jul, 53(7), 479 - 511
{Antipseudomonal activity of carbapenem antibiotics}; Sunagawa M et al.; To date, three carbapenem antibiotics have been introduced for clinical use, and they can be structurally classified into two types . One is a natural type that has the naturally-occurring carbapenem skeleton and a strongly basic (cationic) moiety in the C-2 side chain, like imipenem or panipenem . The other is a new generation carbapenem, meropenem, which has the 1 beta-methyl carbapenem skeleton and a less basic group in the C-2 side chain . It was reported that there were some significant differences among these two types of carbapenems concerning the antimicrobial profile, especially the antipseudomonal activity . Since Pseudomonas aeruginosa was one of the target pathogens of carbapenem antibiotics, these facts prompted us to overview the different mode of action among imipenem, panipenem and meropenem and clarify the structure-activity relationships of carbapenems with regard to the antipseudomonal activities . In this article, we discuss that both the chemical structure and the physicochemical properties of carbapenems greatly influence a variety of antipsedomonal actions including MIC, affinity for PBPs, outer membrane permeability, interaction with various beta-lactamases and multidrug efflux systems etc., and that the cationic center in the C-2 side chain plays an important role in antipseudomonal activities . This review will be helpful in developing new types of antipseudomonal carbapenems and/or new clinical applications of carbapenem antibiotics for treating pseudomonal infection.

J Microbiol Methods, 2000 Oct, 42(2), 149 - 58
Rapid spectrophotometric determination of 2,4,6-trinitrotoluene in a Pseudomonas enzyme assay; Oh B et al.; Although TNT (2,4,6-trinitrotoluene) and its degradation products can be quantified by HPLC, this method is not suitable for simultaneous analyses of the numerous samples typically encountered in enzyme studies . To solve this problem, we developed a simple and rapid spectrophotometric assay for TNT and tested the procedure using partially purified nitroreductase(s) from a Pseudomonas aeruginosa isolate, which transformed TNT in the culture medium . In highly alkaline solution, TNT (pK(a)=11.99) exhibits significant absorbance at 447 nm, while major metabolites, 2-amino-4, 6-dinitrotoluene (2ADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and 2,6-diamino-4-nitrotoluene (2,6DANT) display no absorbance at this wavelength . Assay mixtures of TNT, Tris-HCl buffer, a reductant, and the enzyme(s) were analyzed by measuring absorbance 4 min after adjusting the pH to 12.2 . TNT transformation to colorless metabolites was linear with respect to protein and substrate concentrations . Using the assay, we determined that TNT nitroreductase(s) from the isolate required an electron donor and preferred NADH to NADPH . TNT transformation increased when NAD was recycled to NADH using glucose-6-phosphate (GP) and glucose-6-phosphate dehydrogenase (GPDH) . Enzymatic transformation of TNT was completely inhibited by Cu(2+) (5 mM) and was partially inhibited by other divalent metallic cations . Because the assay is sensitive to ammonium sulfate, dithiothreitol, ascorbic acid, and sodium phosphate, extracts should be assayed in the absence of these components.

Clin Infect Dis, 2000 Sep, 31(3), 705 - 11 Epub 2000 Oct 04.
Bacteremia due to Stenotrophomonas maltophilia in patients with hematologic malignancies; Micozzi A et al.; Predisposing factors, clinical characteristics, and antimicrobial treatment of 37 hematology patients with Stenotrophomonas maltophilia bacteremia who were seen at the department of hematology of the University La Sapienza (Rome) from 1987 to 1996 were evaluated . The results were compared with a control group of patients with Pseudomonas aeruginosa bacteremia . Profound neutropenia was more prolonged in the S . maltophilia group (P=.025), severe cellulitis occurred only in S . maltophilia-infected patients (11 {30%}; P=.0002), and the bacteremia presented as breakthrough infection in 56% of the cases due to S . maltophilia (vs . only 24% of those due to P . aeruginosa; P=.002) . Acute mortality rates associated with S . maltophilia and P . aeruginosa bacteremia were 24% and 21%, respectively . In both groups, profound neutropenia and hypotension at the onset of bacteremia, duration of profound neutropenia during bacteremia, severity-of-illness score > or =4, and inappropriate antibacterial treatment were factors significantly associated with death . Most S . maltophilia isolates were resistant to aminoglycosides, beta-lactams, and ciprofloxacin . Cotrimoxazole and ticarcillin-clavulanic acid showed borderline activity . Prompt administration of in vitro-active antibiotics may improve the prognosis of S . maltophilia bacteremia, especially for immunocompromised patients, and novel drug combinations are needed for the treatment of severe infections.

Nat Struct Biol, 2000 Oct, 7(10), 918 - 25
Crystal structure of the class D beta-lactamase OXA-10; Paetzel M et al.; We report the crystal structure of a class D beta-lactamase, the broad spectrum enzyme OXA-10 from Pseudomonas aeruginosa at 2.0 A resolution . There are significant differences between the overall fold observed in this structure and those of the evolutionarily related class A and class C beta-lactamases . Furthermore, the structure suggests the unique, cation mediated formation of a homodimer . Kinetic and hydrodynamic data shows that the dimer is a relevant species in solution and is the more active form of the enzyme . Comparison of the molecular details of the active sites of the class A and class C enzymes with the OXA-10 structure reveals that there is no counterpart in OXA-10 to the residues proposed to act as general bases in either of these enzymes (Glu 166 and Tyr 150, respectively) . Our structures of the native and chloride inhibited forms of OXA-10 suggest that the class D enzymes have evolved a distinct catalytic mechanism for beta-lactam hydrolysis . Clinical variants of OXA-10 are also discussed in light of the structure.

Anesteziol Reanimatol, 2000 Jul-Aug, (4), 54 - 6
{Nosocomial pneumonia in patients with severe craniocerebral trauma in intensive care units}; Shatvorian BR et al.; Eighty-three patients with severe craniocerebral injuries (CCI) were treated at Institute of Neurosurgery in 1999 . Pulmonary infectious complications occurred in 16 of 25 patients with severe CCI . Early nosocomial pneumonia (NP) was diagnosed in 18% and the so-called late NP (associated with artificial ventilation of the lungs) in 35% . Coma longer than 4 days increased the incidence of NP to 62% . The main pathogens of NP are gram-negative aerobic bacteria (61%), the predominant agent being Pseudomonas aeruginosa (18.9%) . 76% isolated microorganisms were multiresistant . The most significant risk factors as regards NP in patients with severe CCI were coma combined with bulbar and pseudobulbar disorders.

Immunology, 2000 Oct, 101(2), 271 - 8
Human endothelial cells are activated by interferon-gamma plus tumour necrosis factor-alpha to kill intracellular Pseudomonas aeruginosa; De Assis MC et al.; Proinflammatory cytokines have been shown to activate endothelial cells . To investigate the effect of cytokines on the interaction of human umbilical vein endothelial cells (HUVEC) with Pseudomonas aeruginosa, cells were treated with interferon-gamma (IFN-gamma) plus tumour necrosis factor-alpha (TNF-alpha) for 24 hr and exposed to P . aeruginosa suspension for 1 hr . Light microscopy showed that activated cells internalized significantly more bacteria than control cells . To ascertain the effect of cytokines on the microbicidal activity of HUVEC, the concentrations of viable intracellular (IC) bacteria in control and activated cells were determined, at 1 and 5 hr postinfection, by the gentamicin exclusion assay . In control cells, no significant decrease in the concentration of bacteria was detected 5 hr postinfection . In contrast, in activated cells the concentration of viable bacteria at 5 hr was significantly lower . Concentrations of superoxide and hydrogen peroxide detected in supernatants of activated cells were significantly higher than in control cell supernatants . HUVEC anti-P . aeruginosa activity was insensitive to the antioxidants superoxide dismutase, dimethylthiourea and allopurinol as well as to the L-arginine analogues aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), but was significantly inhibited by catalase . Our results indicate that HUVEC can be activated by IFN-gamma plus TNF-alpha to kill IC P . aeruginosa and suggest a role for reactive oxygen radicals, notably hydrogen peroxide, in HUVEC antibacterial activity.

Clin Exp Immunol, 2000 Oct, 122(1), 67 - 71
Intrapulmonary concentrations of inflammatory cytokines in a mouse model of chronic respiratory infection caused by Pseudomonas aeruginosa; Yanagihara K et al.; We investigated the role of inflammatory cytokines in a mouse model of chronic Pseudomonas aeruginosa infection mimicking diffuse panbronchiolitis (DPB), and determined the effects of clarithromycin therapy on the production of these cytokines . The concentrations of IL-1beta, IL-2, IL-4, IL-5, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) were measured serially in the lungs of mice with experimentally induced chronic respiratory P . aeruginosa infection until 60 days after inoculation . The concentrations of these cytokines during the course of the disease were significantly higher than baseline (before inoculation, P<0.01 for all cytokines) . Clarithromycin significantly inhibited the production of IL-1beta and TNF-alpha in the lung (P<0.01) . The same treatment also reduced the levels of other cytokines, albeit insignificantly . Treatment with anti-TNF-alpha antibody significantly reduced the number of pulmonary lymphocytes and concentration of IL-1beta in the lung (P<0.01), but did not change the number of viable bacteria . Our findings resemble those detected in bronchoalveolar lavage fluid of patients with DPB and indicate that inflammatory cytokines play an important role in chronic P . aeruginosa lung infection . Our results also show that macrolides modulated the production of these cytokines, ultimately reducing lymphocyte accumulation in the lung . Our data suggest that anti-TNF-alpha antibody might be a useful new strategy for the treatment of chronic respiratory P . aeruginosa infection.

Laryngorhinootologie, 2000 Jul, 79(7), 400 - 3
{hBD-2 gene expression in nasal mucosa}; Meyer JE et al.; BACKGROUND: Chronic sinusitis is one of the frequent inflammatory diseases and has a complex pathogenesis . A substantial factor seems to be recurrent bacterial infections . Pseudomonas aeruginosa (PA) frequently can be found in nasal smears of patients with persistent sinus symptoms after sinus surgery . Lately a new antimicrobial peptide of epithelial origin, human Beta-Defensin-2 (hBD-2), with a strong antibacterial effect against PA could be identified within lesional skin scales of patients suffering psoriasis . Aim of this study was to investigate hBD-2-mRNA expression in nasal cells and tissue . METHODS: Total RNA was extracted from nasal polyps and turbinates following TRIzol protocol . Epithelial cells and fibroblasts of nasal human tissue were isolated and cultivated . The cells were stimulated with PA using different time points and different concentrations . Total RNA was isolated as mentioned above, reverse transcribed and amplified in a Semi-quantitative Reverse Transcriptase PCR (SQRT-PCR) with genespecific hBD-2 primers . RESULTS: PA induces time- and dose-dependently hBD-2 gene expression in nasal epithelial cells . Unstimulated epithelial nasal cells were able to express hBD-2 mRNA constitutively, whereas nasal fibroblasts showed no hBD-2 mRNA expression . Nasal polyps showed a comparable less hBD-2 gene expression then nasal turbinates . CONCLUSIONS: hBD-2 possibly mediates a specific, early starting antimicrobial defense strategy of the nasal mucosa . This hypothesis would explain persistent infections with PA through diminished hBD-2 gene expression.

Pneumonol Alergol Pol, 2000, 68(3-4), 101 - 8
{Pseudomonas aerogunosa pneumonia in patients treated at the Hospital for Chest Diseases}; Graczyk J et al.; Retrospective analysis of pneumonia caused by Pseudomonas aeruginosa was made in 66 patients, treated in hospital . Nosocomial pneumonia was diagnosed in 11 (17%) patients . In 51 patients coexisting lung diseases were present: mainly COPD and bronchiectasis . Strains of Pseudomonas aeruginosa were susceptible mostly to imipenem, meropenem, aztreonam, ticarcillin-clavulanic acid, ceftazidime, ciprofloxacin, amikacin, piperacillin-tazobactam, netilmicin . Duration of treatment in hospital was very long--59% were treated over 30 days . Combined antibacterial therapy was applied in 35 (53%) patients and monotherapy, often with different antibiotics--in 31 (47%) patients . Treatment was successful in 45 (68%) patients . In 9 patients the results of treatment was not successful: mainly because of empyema in 7 pts . Twelve (18%) patients (with coexisting COPD--6 and lung cancer--6) died . We can support current recommendations for treatment of Pseudomonas aeruginosa infection with combination of aminoglycosides or fluoroquinolones plus one of remaining antipseudomonal antibiotics . Treatment failures occurred mainly in patients with severe coexisting diseases and/or empyema.

Biochim Biophys Acta, 2000 Jun 15, 1479(1-2), 265 - 74
Purification, physico-chemical characterization and sequence of a heat labile alkaline metalloprotease isolated from a psychrophilic Pseudomonas species; Chessa JP et al.; The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated from Antarctica has been purified and characterized . The gene encoding PAP has been cloned and sequenced and the derived amino acid sequence shows 66% identity with the mesophilic alkaline metalloprotease from Pseudomonas aeruginosa IFO 3455 (AP) . Compared to the purified AP, PAP is three times more active at 20 degrees C, is very sensitive to chelating agents and is rapidly inactivated at 45 degrees C . The lower thermostability of PAP can tentatively be explained by a loss of a stabilizing Ca(2+), a decrease in the content of hydrophobic residues and a smaller aliphatic index.

J Bacteriol, 2000 Oct, 182(20), 5925 - 30
On the architecture of the gram-negative bacterial murein sacculus; Pink D et al.; The peptidoglycan network of the murein sacculus must be porous so that nutrients, waste products, and secreted proteins can pass through . Using Escherichia coli and Pseudomonas aeruginosa as a baseline for gram-negative sacculi, the hole size distribution in the peptidoglycan network has been modeled by computer simulation to deduce the network's properties . By requiring that the distribution of glycan chain lengths predicted by the model be in accord with the distribution observed, we conclude that the holes are slits running essentially perpendicular to the local axis of the glycan chains (i . e., the slits run along the long axis of the cell) . This result is in accord with previous permeability measurements of Beveridge and Jack and Demchik and Koch . We outline possible advantages that might accrue to the bacterium via this architecture and suggest ways in which such defect structures might be detected . Certainly, large molecules do penetrate the peptidoglycan layer of gram-negative bacteria, and the small slits that we suggest might be made larger by the bacterium.

J Bacteriol, 2000 Oct, 182(20), 5793 - 8
Myxococcus xanthus dif genes are required for biogenesis of cell surface fibrils essential for social gliding motility; Yang Z et al.; Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes . In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M . xanthus S motility . We have demonstrated here that M . xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili . Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials . Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M . xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion . Genetic studies indicate that the dif genes are linked to the M . xanthus dsp region, a locus known to be crucial for M . xanthus fibril biogenesis and S gliding.

J Trauma, 2000 Sep, 49(3), 511 - 4
Effectiveness of electrolyzed oxidized water irrigation in a burn-wound infection model; Nakae H et al.; OBJECTIVE: The purpose of the study was to determine whether electrolyzed oxidized water (EOW) functions as a bactericide in burn injury with Pseudomonas aeruginosa infection in a rat burn-wound model . METHODS: Anesthetized Sprague-Dawley rats (n = 31) were subjected to third-degree burns to 30% of total body surface area . Two days after injury, all rats were infected with P . aeruginosa using 1 mL of a suspension containing 1 x 10(8) colony-forming units . Rats were assigned to one of three groups: no irrigation (group I), irrigation with physiologic saline (group II), or irrigation with EOW (group III) . Blood culture, endotoxin levels, and survival rates were determined . RESULTS: Survival rate was significantly higher in group III than in groups I or II (p < 0.0001) . Serum endotoxin levels on day 3 after infection in group III were significantly lower than the levels in group I (p < 0.01) and group II (p < 0.01) . There were significant differences between the three groups in the culture of P . aeruginosa (p < 0.05) . CONCLUSION: Irrigation and disinfection with EOW may become useful in preventing burn-wound sepsis.

Bioorg Med Chem, 2000 Aug, 8(8), 1969 - 82
Discovery of novel trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenems; Imamura H et al.; Novel trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenems were designed and synthesized to provide J-111,347 (1a) as the first example of an exceptionally broad-spectrum antibiotic, showing activity against methicillin-resistant Staphyloccocus aureus (MRSA) as well as Pseudomonas aeruginosa . Further derivation of 1a afforded J-111,225 (2a), J-114,870 (3a), and J-114,871 (3b) . which showed improved safety profiles and retained broad-spectrum antibacterial activities.

J Biotechnol, 2000 Sep 29, 83(1-2), 3 - 12
Immunogenic efficacy of differently produced recombinant vaccines candidates against Pseudomonas aeruginosa infections; von Specht BU et al.; Three different variants of the recombinant hybrid outer membrane protein OprF (aa 190-342)-OprI (aa 21-83) could be obtained in high yield after expression in Escherichia coli . The hybrid protein was modified N terminally, either with a minimal histidine tag or with a homologous sequence of OprF . Both recombinant proteins were purified by nickel chelate affinity chromatography under native and denaturing conditions, and this produced three suitable candidates for a vaccination trial, protein His-F-I, which was purified in its native as well as in its refolded form; and the native purified N terminally extended protein, ex-F-I . In mice, significantly higher antibody titers and survival rates after challenge with Pseudomonas aeruginosa were observed following immunization with protein His-F-I, purified under native conditions.

Ann Surg, 2000 Oct, 232(4), 480 - 9
Gut-derived sepsis occurs when the right pathogen with the right virulence genes meets the right host: evidence for in vivo virulence expression in Pseudomonas aeruginosa; Alverdy J et al.; OBJECTIVE: To define the putative role of the PA-I lectin/adhesin, a binding protein of Pseudomonas aeruginosa, on lethal gut-derived sepsis after surgical stress, and to determine if this protein is expressed in vivo in response to physical and chemical changes in the local microenvironment of the intestinal tract after surgical stress . SUMMARY BACKGROUND DATA: Previous work from the authors' laboratory has established that lethal gut-derived sepsis can be induced after the introduction of P . aeruginosa into the cecum of mice after a 30% hepatectomy . This effect does not occur when P . aeruginosa is introduced into the cecum of sham operated control mice . Previous experiments further established that the mechanism of this effect is due to the presence of the PA-I lectin/adhesin of P . aeruginosa, which induces a permeability defect to a lethal cytotoxin of P . aeruginosa, exotoxin A . METHODS: Three strains of P . aeruginosa, one lacking functional PA-I, were tested in two complementary systems to assess virulence . Strains were tested for their ability to adhere to and alter the permeability of cultured human colon epithelial cells, and for their ability to induce mortality when injected into the cecum of mice after a 30% hepatectomy . To determine if PA-I is "in vivo expressed" when present in the cecal environment after hepatectomy, strains were retrieved from the cecum of sham-operated and hepatectomy-treated mice 24 and 48 hours after their introduction into the cecum and their PA-I expression was assessed . RESULTS: Results indicated that PA-I plays a putative role in lethal gut-derived sepsis in the mouse, because strains lacking functional PA-I had an attenuated effect on cultured human epithelial cells, and were nonlethal when injected into the cecum of mice after 30% surgical hepatectomy . Furthermore, surgical stress in the form of hepatectomy significantly altered the intestinal microenvironment, resulting in an increase in luminal norepinephrine associated with an increase in PA-I expression in retrieved strains of P . aeruginosa . Co-incubation of P . aeruginosa with norepinephrine increased PA-I expression in vitro, suggesting that norepinephrine plays a role in the observed response in vivo . CONCLUSIONS: Lethal gut-derived sepsis may occur when intestinal pathogens express virulence determinants in response to environmental signals indicating host stress . In this regard, the PA-I lectin/adhesin of P . aeruginosa appears to be a specific example of in vivo virulence expression in colonizing pathogens in the intestinal tract in response to surgical stress.

Am J Med, 2000 Sep, 109(4), 288 - 95
Association between airway bacterial load and markers of airway inflammation in patients with stable chronic bronchitis; Hill AT et al.; PURPOSE: Viable bacteria are often isolated from airway secretions in clinically stable patients with chronic bronchitis . We hypothesized that the number of organisms and bacterial species might be important modulators of airway inflammation . SUBJECTS AND METHODS: We performed quantitative sputum cultures in 160 stable patients {55 with chronic obstructive pulmonary disease (COPD) and normal serum alpha(1)-antitrypsin levels, 62 with COPD and severe alpha(1)-antitrypsin deficiency (PiZ), and 43 with idiopathic bronchiectasis} . The results were related to several indicators of the mechanisms and severity of airway inflammation . RESULTS: Airway bacterial load correlated with sputum myeloperoxidase level, an indirect measure of neutrophil activation and number (r = 0.50, P<0 . 001); sputum neutrophil chemoattractants {interleukin-8 level (r = 0 . 68, P<0.001) and leukotriene B4 level (r = 0.53, P<0.001)}; sputum leukocyte elastase activity (r = 0.55, P<0.001); and albumin leakage from serum to sputum (r = 0.26, P<0.01) . Markers of inflammation increased at bacterial loads of 10(6) to 10(7) colony-forming units per milliliter, and increased progressively with increasing bacterial load . For example, the median (interquartile range) sputum myeloperoxidase level was 0.3 U/mL (0.1 to 0.5 U/mL) for patients who were not colonized or who had mixed normal oropharyngeal flora alone; 0.5 U/mL (0.2 to 0.7 U/mL) for patients with 10(5) to 10(6) colony-forming units per milliliter (P = 0.07); 0.5 U/mL (0.3 to 1.2 U/mL) for patients with 10(6) to 10(7) colony-forming units per milliliter (P<0.01); 0.7 U/mL (0.3 to 1.2 U/mL) for patients with 10(7) to 10(8) colony-forming units per milliliter (P <0.005); and 2.4 U/mL (0.7 to 4.8 U/mL) for patients with 10(8) or greater colony-forming units per milliliter (P<0.0001) . The bacterial species influenced airway inflammation; for example, sputum myeloperoxidase activity was greater (P<0.005) in patients colonized with Pseudomonas aeruginosa {median 32 U/mL (interquartile range, 20 to 65 U/mL)} than those colonized with nontypeable Hemophilus influenzae {4 U/mL (2 to 31 U/mL)}, which in turn was greater (P = 0.01) than among those colonized with Moraxella catarrhalis {1.1 U/mL (0.6 to 1.8 U/mL)} . We did not find a relation between bacterial load and lung function.CONCLUSIONS: The bacterial load and species contribute to airway inflammation in patients with stable chronic bronchitis . Further studies are required to determine the consequences of bacterial colonization on patient morbidity and decline in lung function.

Immunol Lett, 2000 Oct 3, 74(2), 165 - 72
Synergism of Pseudomonas aeruginosa exotoxin A with endotoxin, superantigen, or TNF results in TNFR1- and TNFR2-dependent liver toxicity in mice; Schumann J et al.; Pseudomonas aeruginosa is a potentially dangerous Gram-negative nosocomial pathogen, causing bacteremia in debilitated patients, and a prominent cause of bacterial cholangitis . Opportunistic infections with other nosocomial pathogens, e.g . Staphylococcus aureus, are common . Hence, multi-intoxication with P . aeruginosa exotoxin A (PEA) and other bacterial toxins, including endotoxin (LPS) and the superantigen S . aureus enterotoxin B (SEB), is very likely . Here we show that PEA synergistically interacted with LPS, SEB, or recombinant murine tumor necrosis factor alpha (rmuTNF) in mice, resulting in severe liver injury . Enhanced and prolonged circulation of cytokines, including TNF, which depended on the presence of T cells, was a remarkable feature of synergistic PEA/LPS- or PEA/SEB-induced hepatotoxicity . PEA/LPS-, PEA/SEB- or PEA/rmuTNF-induced liver injury was mediated by both TNF receptors (TNFRs), i.e . TNFR1 and TNFR2 . In view of the fact that TNFR1, but not TNFR2, signaling is unequivocally required for host defense, our results suggest that anti-TNFR2 strategies might be beneficial to protect the liver from inflammatory damage caused by synergistic interactions of PEA with other TNF-inducing bacterial toxins.

Mol Genet Metab, 2000 Aug, 70(4), 316 - 21
A novel CFTR frame-shift mutation, 935delA, in two Hispanic cystic fibrosis patients; Wang J et al.; The currently available mutation analysis panel detects about 50-60% of CFTR mutations in Hispanic patients . In order to search for Hispanic CF mutations, we developed a temporal temperature gradient gel electrophoresis (TTGE) method to screen for unknown mutations . Using TTGE to study the CFTR gene has lead to the discovery of many novel mutations in Hispanic patients . A novel frame-shift mutation, 935delA, was found in two unrelated patients . One was heterozygous for two novel frame-shift mutations, 663delT and 935delA, and the other was heterozygous for DeltaF508 and 935delA . Both patients showed severe phenotype with meconium ileus, pancreatic insufficiency, and early pulmonary microbial colonization with Pseudomonas aeruginosa . Patient 1 died at 4 years of age . Patient 2 had an upper lobectomy . The 935delA mutation produces a truncated polypeptide with only 21% of the full-length protein . The severe course of clinical manifestation is consistent with two oppressively truncated mutant polypeptides encoded by both mutant alleles in patient 1 and the compound heterozygosity truncation and DeltaF508 mutations in patient 2 .

Chem Pharm Bull (Tokyo), 2000 Sep, 48(9), 1286 - 92
Phenolic constituents of licorice . VIII . Structures of glicophenone and glicoisoflavanone, and effects of licorice phenolics on methicillin-resistant Staphylococcus aureus; Hatano T et al.; Two new phenolic compounds, glicophenone (1) and glicoisoflavanone (2), were isolated from commercial licorice, and their structures were elucidated on the basis of spectroscopic data . Antibacterial assays of licorice phenolics for Staphylococcus aureus, including four strains of methicillin-resistant S . aureus (MRSA), and also for Escherichia coli K12 and Pseudomonas aeruginosa PAO1, were then examined . Two compounds among them, 8-(gamma,gamma-dimethylallyl)-wighteone (21) and 3'-(gamma,gamma-dimethylallyl)-kievitone (28), showed remarkable antibacterial effects {minimum inhibitory concentrations (MICs), 8 microg/ml on the MRSA strains and methicillin-sensitive S . aureus . Licochalcone A (14), gancaonin G (20), isoangustone A (24), glyasperins C (30) and D (31), glabridin, (32), licoricidin (33), glycycoumarin (34) and licocoumarone (40) showed antibacterial effects on the MRSA strains with MIC values of 16 microg/ml . Effects on the beta-lactam resistance of the MRSA strains were also examined, and licoricidin (33) noticeably decreased the resistance of the MRSA strains against oxacillin, as shown by the reduction in the MICs of oxacillin (lower than 1/128-1/1000 in the presence of 8 microg/ml of 33, and 1/8-1/32 in the presence of 4 microg/ml of 33) . Mechanistic study suggested that 33 does not inhibit the formation of penicillin-binding protein 2' (PBP2'), but affects the enzymatic function of PBP2'.

Infect Immun, 2000 Oct, 68(10), 6066 - 8
Pseudomonas aeruginosa ExoT is a Rho GTPase-activating protein; Krall R et al.; Transient intracellular expression of ExoT in CHO cells stimulated cell rounding and actin reorganization . Biochemical studies showed that ExoT was a GTPase-activating protein for RhoA, Rac1, and Cdc42 . Together, these data show that ExoT interferes with Rho signal transduction pathways, which regulate actin organization, exocytosis, cell cycle progression, and phagocytosis.

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2861 - 4
Two efflux systems expressed simultaneously in multidrug-resistant Pseudomonas aeruginosa; Pumbwe L et al.; Simultaneous overexpression of MexAB-OprM and MexEF-OprN was demonstrated for a multiply antibiotic-resistant clinical isolate of Pseudomonas aeruginosa (G49) . G49 also had decreased expression of OprF . No mutations in mexR or its upstream promoter region, mexT, oprM, oprF, or sigX were revealed, suggesting regulation by a hitherto undescribed locus.

Antibiot Khimioter, 2000, 45(8), 17 - 20
{Antibiotics sensitivity and characteristics of the esculin-positive Pseudomonas aeruginosa biovar}; Sivolodskii EP; Strains of Pseudomonas aeruginosa hydrolyzing esculin were isolated for the first time . They amount to 17.1 +/- 2.0% (60 from 325) of the investigated P . aeruginosa strains isolated from the clinical material in St . Petersburg . Esculin hydrolysis was measured by micromethod in plates, results were analysed after 3-hours incubation at 37 degrees C . Esculin-positive strains possesed biovar properties: they are widely spread, demonstrated other characteristic features (absence of triethylamine odour, specific colonies lysis), are stable on ability to hydrolyse esculin while culture storage and after repeated culturing . Typical strain of esculinolytica biovar was deposited into the culture collection of the National Research Institute of Agricultural Microbiology as P . aeruginosa ARRIAM 64-A . Susceptibility testing of the esculin-positive strains by disk-diffusion method revealed that most strains were inhibited by imipenem (86.6%), amikacin (75.0%), ceftazidime (65.0%), meropenem (60.0%), aztreonam (51.6%) . The percent of strains susceptible to other antibiotics was lower: azlocillin--33.3%, netilmycin--33.3%, piperacillin--26.6%, ceftriaxon--18.3% . Only small number of strains were inhibited by ciprofloxacin (8.3%), gentamycin (3.4%), cefoperazone (1.7%) and carbenicillin (1.7%) . The results may be used for empiric therapy before the isolated strain susceptibility is tested but only according to positive esculin-hydrolysis express-test evaluated in 3-hours period.

Antibiot Khimioter, 2000, 45(7), 14 - 6
{The pharmacodynamics and pharmacokinetics of fluoroquinolones in the evaluation of antibacterial therapy regimens}; Smirnova LB et al.; Sensitivity of 505 strains of gram-negative and gram-positive microorganisms to II generation fluoroquinolones (ciprofloxacin, pefloxacin) was determined . Strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus with different level of sensitivity were selected . Pharmacokinetics of the drugs was investigated after their administration per os in one dose . The resulting indices were used for calculation of the following parameters--Cmax/MIC and AUC/MIC . These parameters may be used in evaluation of the drugs efficacy and for dosing corrections.

Nature, 2000 Aug 31, 406(6799), 959 - 64
Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen; Stover CK et al.; Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections . A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants . Here we report the complete sequence of P . aeruginosa strain PAO1 . At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P . aeruginosa . Consistent with its larger genome size and environmental adaptability, P . aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems . We propose that the size and complexity of the P . aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

Clin Experiment Ophthalmol, 2000 Jun, 28(3), 197 - 200
Pro-inflammatory cytokine/chemokine gene expression in human corneal epithelial cells colonized by Pseudomonas aeruginosa; Xue ML et al.; Pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-1beta (IL- 1beta), tumour necrosis factor-alpha (TNF-alpha) and chemokine interleukin-8 (IL-8) may play a significant role in the regulation of bacterial corneal infection . The aim of this study was to investigate the gene expression of these four mediators in a human corneal epithelial cell line challenged with Pseudomonas aeruginosa within 12 h . Human corneal epithelial monolayers were colonized with P . aeruginosa strains Paerl, 6206 and 6294 . Expression of IL-6, IL-8, IL-1beta and TNF-alpha was analysed using semi-quantitative reverse transcription-polymerase chain reaction . Results showed that both IL-6 and IL-8 mRNA were expressed very early (4 h) during bacterial colonization and remained at high levels until the end of the experiment . Expression of IL-1beta and TNF-alpha mRNA appeared at 8 h after bacterial stimulation . No expression of IL-8, IL-1beta and TNF-alpha mRNA was observed in unstimulated cells . Interleukin-6 mRNA was expressed at low levels in unstimulated cells . In conclusion, bacterial colonization of human corneal epithelial cells induced expression of IL-6 and IL-8 mRNA earlier and at higher levels than IL-1beta and TNF-alpha mRNA.

Clin Experiment Ophthalmol, 2000 Jun, 28(3), 191 - 3
Co-incubation of Acanthamoeba castellanii with strains of Pseudomonas aeruginosa alters the survival of amoeba; Cengiz AM et al.; Enhanced survival of Acanthamoeba castellanii has previously been reported following co-incubation with a single strain of Pseudomonas aeruginosa . The aim of this study was to evaluate the impact of different strains of P . aeruginosa on amoebae survival . Four contact lens solutions were challenged with A . castellanii for between 6 and 24 h, and survival rates of amoeba were calculated . Subsequently, A . castellanii was co-incubated with different strains of P . aeruginosa (strain 6294, an invasive isolate; 6206, a cytotoxic isolate; and Paer 001, a null isolate) . Differences in amoeba survival over time between solutions for each bacterial strain were analysed . Non-neutralized hydrogen peroxide was the most effective system against A . castellani at all time points (P<0.05) . Survival rates were not different between multipurpose solutions and neutralized hydrogen peroxide . Co-incubation with P . aeruginosa altered amoeba survival, and maximum survival occurred in the presence of the invasive strain of P . aeruginosa . Enhanced amoeba survival may occur in the presence of certain strains of Gram-negative bacteria, and with certain types of contact lens disinfection systems.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 141 - 6
Genomics of the 35-kb pvd locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa; Lehoux DE et al.; Novel putative pyoverdine synthetase pvdIJK genes were found upstream of pvdD in the 6.2-Mb chromosome of Pseudomonas aerugilosa strain PAO1 . These genes formed a locus implicated in pyoverdine biosynthesis . Sequence analysis showed that the product of these genes shared 43%, 60% and 57% identity with PvdD . PvdIJK are thought to be implicated in synthesis of pyoverdine, a siderophore chelating Fe3+ . A pvdI mutant was obtained by gene disruption mutagenesis and confirmed by Southern hybridization . The pvdl mutant produced gave no significant growth on solid media supplemented with the iron chelator 2,2-dipyridyl; while the PvdI- phenotype abolished pyoverdine fluorescence . The role of PvdI in pathogenicity was tested by measuring the in vivo growth of P . aeruginosa wild-type and mutant strains in a chronic lung infection rat model, and by measuring the competitive infectivity index into a neutropenic mice model . The data obtained confirmed the importance of PvdI in virulence and iron uptake.

Ann Pharmacother, 2000 Sep, 34(9), 1017 - 9
Alatrofloxacin-induced seizures during slow intravenous infusion; Melvani S et al.; OBJECTIVE: To report a case of seizures associated with slow infusion (1-2 h) of alatrofloxacin, the prodrug of trovafloxacin . CASE SUMMARY: A 37-year-old Asian man was admitted to the hospital for a distal pancreatectomy and drainage of a pseudocyst . Postoperative complications developed, which included peritonitis and pneumonia, requiring intensive care admission . Cultures from peritoneal drainage fluid and sputum isolated Klebsiella pneumoniae and Pseudomonas aeruginosa, respectively . He was treated with multiple courses of antibiotics, including intravenous gentamicin, metronidazole, vancomycin, meropenem, and ceftazidime . After three weeks, the patient still had sepsis and began therapy with alatrofloxacin in addition to ceftazidime and vancomycin . Alatrofloxacin infusion was administered according to product information instructions . Fifteen minutes after the first dose was started, the patient developed generalized clonus . On rechallenge, infusing at half the initial rate, the seizure recurred; consequently, the infusion was discontinued and replaced with intravenous ciprofloxacin and metronidazole . The patient remained seizure free thereafter . DISCUSSION: Fluoroquinolones have been implicated in central nervous system adverse effects, including seizures, which have been reported with other fluoroquinolones but not with alatrofloxacin or trovafloxacin . In these reports, the patients often had preexisting risk factors such as increased age and electrolyte imbalances . The only apparent predisposition in this patient was mild hyponatremia . CONCLUSIONS: Alatrofloxacin may cause seizures even during slow infusion . This case highlights the need for caution when commencing parenteral fluoroquinolone therapy, particularly with a new agent.

Chem Biol, 2000 Sep, 7(9), 709 - 18
Directed evolution of an enantioselective lipase; Liebeton K et al.; BACKGROUND: The biocatalytic production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industry . In most cases, however, it is impossible to identify an enzyme that possesses the desired enantioselectivity . Therefore, there is a strong need to create by molecular biological methods novel enzymes which display high enantioselectivity . RESULTS: A bacterial lipase from Pseudomonas aeruginosa (PAL) was evolved to catalyze with high enantioselectivity the hydrolysis of the chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester . Successive rounds of random mutagenesis by ep-PCR and saturation mutagenesis resulted in an increase in enantioselectivity from E=1.1 for the wild-type enzyme to E=25.8 for the best variant which carried five amino acid substitutions . The recently solved three-dimensional structure of PAL allowed us to analyze the structural consequences of these substitutions . CONCLUSIONS: A highly enantioselective lipase was created by increasing the flexibility of distinct loops of the enzyme . Our results demonstrate that enantioselective enzymes can be created by directed evolution, thereby opening up a large area of novel applications in biotechnology.

J Antimicrob Chemother, 2000 Sep, 46(3), 377 - 84
Specific phospholipids enhance the activity of beta-lactam antibiotics against Pseudomonas aeruginosa; Krogfelt KA et al.; Pseudomonas aeruginosa PAO1 became considerably more sensitive to the action of ampicillin when grown in the presence of certain phospholipids . Only phospholipids capable of forming lipid bilayers or micelles proved to be capable of enhancing ampicillin activity . Of the phospholipids tested, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate, also called monopalmitoylphosphatidic acid (MPPA), was the best enhancer . In the absence of MPPA, the MIC and MBC of ampicillin for P . aeruginosa PAO1 were 1 and 2 g/L, respectively . In the presence of MPPA, the MIC and MBC were 20 and 40 mg/L, respectively . MPPA was shown to enhance ampicillin activity by binding both Ca(2+) and Mg(2+), suggesting that the mechanism of enhancement is similar to that previously reported for Ca(2+) and Mg(2+) chelators . Surprisingly, MPPA by itself slowed the growth of four mucoid multiply antibiotic-resistant strains of P . aeruginosa recently isolated from the sputum of cystic fibrosis patients, and enhanced their sensitivity to piperacillin . It also increased the sensitivity of two ceftazidime-resistant P . aeruginosa cystic fibrosis strains to ceftazidime.

Curr Microbiol, 2000 Oct, 41(4), 290 - 4
Characterization of the alanine racemases from Pseudomonas aeruginosa PAO1; Strych U et al.; Alanine racemases are ubiquitous, almost uniquely prokaryotic enzymes catalyzing the racemization between l- and d-alanine . The requirement for d-alanine as a necessary component of the bacterial cell wall makes this class of enzymes a logical target for the development of novel antibiotics . In an effort to better understand the structure and mechanism of these enzymes, we have cloned the two independent alanine racemases from Pseudomonas aeruginosa, an important opportunistic bacterial pathogen of humans and animals . The dadX(PA) and alr(PA) genes have been sequenced, overexpressed, and their activity was demonstrated by complementing d-alanine auxotrophs of Escherichia coli . Both gene products were purified to electrophoretic homogeneity, the enzymes were characterized biochemically, and preliminary crystals were obtained.

Trans R Soc Trop Med Hyg, 2000 May-Jun, 94(3), 315 - 7
Microbial keratitis--a late complication of penetrating keratoplasty; Huang SC et al.; A retrospective review of 323 penetrating keratoplasties performed in Taiwan between January 1993 and December 1997 revealed that late microbial keratitis developed in 39 eyes of 36 patients (12.1%) . All patients were operated on by the same surgeon, and all were followed for at least 1 year . The mean interval between the corneal transplantation and the onset of graft infection was 8.6 +/- 8.8 months (range 3 weeks-47 months) . Predisposing risk factors for keratitis included chronic blepharitis with poor lid hygiene (43.6%), suture-related problems (38.5%), dry eyes (28.2%), epithelial defects (25.6%), and use of contact lenses (5.1%) . Infectious keratitis was diagnosed within 6 months after keratoplasty in 59% of cases . Positive cultures were obtained in 100% of the ulcers; Pseudomonas aeruginosa and Staphylococcus aureus were the most common pathogens . In the final visual outcome assessment, 30.8% of cases had clear grafts, 20.5% had graft failures, and 10.3% had corneal perforations.

N Engl J Med, 2000 Sep 7, 343(10), 695 - 700
Endemic Pseudomonas aeruginosa infection in a neonatal intensive care unit; Foca M et al.; BACKGROUND: Nosocomial infections due to Pseudomonas aeruginosa have been well described, but the environmental reservoir of the organism varies . We conducted an epidemiologic and molecular investigation of endemic P . aeruginosa infection among infants in a neonatal intensive care unit that was associated with carriage of the organisms on the hands of health care workers . METHODS: In August 1998, colonization or infection with P . aeruginosa was identified in six infants . Surveillance cultures for P . aeruginosa were obtained from the other 27 infants in the unit, and possible environmental reservoirs were also assessed . The hands of health care workers were inspected and cultured, and risk factors for P . aeruginosa colonization were evaluated . Isolates were analyzed for clonality by pulsed-field gel electrophoresis . RESULTS: Surveillance cultures showed that three additional infants were colonized with P . aeruginosa . Cultures of environmental specimens were negative, but cultures of the hands of 10 of 165 health care workers (6 percent) were positive for P . aeruginosa . Increasing age (P=0.05) and a history of the use of artificial fingernails or nail wraps (P=0.03) were both risk factors for colonization of the hands . From January 1997 to August 1998, 49 infants were infected or colonized with P . aeruginosa . Pulsed-field gel electrophoresis demonstrated that 17 of these infants and 1 health care worker who had onychomycosis had the same clone . Infants who were exposed to this health care worker in August 1998 were at greater risk of having this clone than infants who were not exposed to this health care worker (odds ratio, 41.2; 95 percent confidence interval, 1.8 to 940.0; P=0.006) . CONCLUSIONS: An increased rate of infection and colonization with P . aeruginosa among infants in neonatal intensive care units should be investigated by assessing potential reservoirs, including environmental sources as well as patients and health care workers.

Biochem Biophys Res Commun, 2000 Sep 7, 275(3), 854 - 8
Protein delivery by Pseudomonas type III secretion system: Ex vivo complementation of p67(phox)-deficient chronic granulomatous disease; Polack B et al.; Bacterial type III secretion system drives the translocation of virulence factors into the cystosol of host target cells . In phagocytes and in Epstein-Barr virus immortalized B lymphocytes, NADPH oxidase generates O(-2) through an electron transfer chain the activity of which depends on the assembly of three, p67(phox), p47(phox) and p40(phox) cytosolic activating factors with Rac 1/2 and a membrane redox component, cytochrome b(558) . In p67(phox) deficient chronic granulomatous disease (CGD) patients, p67-phox is missing and NADPH oxidase activity is abolished . ExoS is a virulence factor of Pseudomonas aeruginosa which is secreted via the type III secretion system: it was fused with p67(phox) . Pseudomonas aeruginosa synthesized and translocated the hybrid ExoS-p67(phox) fusion protein into the cytosol of B lymphocytes via the type III secretion system . Purified ExoS-p67(phox) hybrid protein was as efficient as normal recombinant p67(phox) in cell-free reconstitution of NADPH oxidase activity . Therefore, ExoS-p67(phox) was transferred via the type III secretion system of Pseudomonas aeruginosa into the cytosol of B lymphocytes from a p67(phox)-deficient CGD patient and functionally reconstituted NADPH oxidase activity . In the complementation process, ExoS acted as a molecular courier for protein delivery: the reconstitution of an active NADPH oxidase complex suggests type III secretion system to be a new approach for cellular therapy .

Mol Microbiol, 2000 Sep, 37(5), 981 - 8
Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts; Mahajan-Miklos S et al.; Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects . Our laboratory has developed a multihost pathogenesis system based on the P . aeruginosa clinical isolate PA14, in which non-mammalian hosts are used to screen directly for virulence-attenuated mutants . The majority of PA14 mutants isolated using non-mammalian hosts also displayed reduced virulence in a burned mouse model . Surprisingly, only a few host-specific virulence factors were identified, and many of the P . aeruginosa mutants were attenuated in virulence in all the hosts . These studies illustrate the extensive conservation in the virulence mechanisms used by P . aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis . Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.

J Appl Microbiol, 2000 Aug, 89(2), 289 - 95
Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1; Winder CL et al.; Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations . The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa . However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure . This experiment induced resistance in cultures of Ps . aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms . The induced resistance was observed as a gradual increase in MIC with each new passage . The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment . The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations . T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide . This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC . The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal . It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT).

Am J Respir Cell Mol Biol, 2000 Sep, 23(3), 304 - 12
Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors; Rajan S et al.; Airway epithelial cells can respond to infection by activating several signaling pathways . We examined the induction of apoptosis in response to Pseudomonas aeruginosa PAO1 in normal cells and several cystic fibrosis (CF) and corrected cell lines . Epithelial cells in monolayers with tight junctions, confirmed by apical ZO-1 staining demonstrated by confocal microscopy, were entirely resistant to PAO1-induced apoptosis . In contrast, cell lines such as 9HTEo(-) cells that do not form tight junctions were susceptible, with 50% of the population apoptotic after 6 h of exposure to PAO1 . CF transmembrane conductance regulator (CFTR) dysfunction caused by different mechanisms (trafficking mutations, overexpression of the regulatory domain or antisense constructs) did not alter rates of apoptosis, nor were differences apparent in terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling detection of apoptotic airway cells from PAO1 infected cftr -/- or control mice . Bacterial expression of specific adhesins, complete lipopolysaccharide, and a functional type III secretion system were all necessary to evoke apoptosis even in susceptible epithelial cells . Unlike other mucosal surfaces, the airway epithelium is highly resistant to apoptosis, and this response is activated only when the appropriate epithelial conditions are present as well as fully virulent P . aeruginosa capable of coordinately expressing both adhesins and cytotoxins.

Mol Cell Probes, 2000 Aug, 14(4), 199 - 204
Rapid identification of Pseudomonas aeruginosa from ocular isolates by PCR using exotoxin A-specific primers; Song KP et al.; The purpose of this research was to evaluate the use of PCR for the identification of ocular isolates of Pseudomonas aeruginosa by using primers specific to the exotoxin A gene of the bacteria . Genomic DNA was obtained from ocular microbial isolates of keratitis patients . Primers were designed based on the published sequence of the exotoxin A gene of P . aeruginosa . Using the primers designed, PCR reactions were performed on the DNA samples . The PCR was also examined for its specificity and sensitivity . In addition, a direct PCR using heating method was attempted on P . aeruginosa with no separate DNA extraction step . ATCC strains of P . aeruginosa were included as positive controls . The rest of the bacteria other than P . aeruginosa served as negative controls . A single band was obtained when analysed on agarose gel electrophoresis only from samples that contained genomic DNA of P . aeruginosa . The direct PCR method was also successful with the same band produced from the amplification . The whole process was completed within 4 h . The direct PCR amplification targeting at the exotoxin A gene of P . aeruginosa is potentially a rapid, specific, sensitive and relatively simple method for the identification of ocular isolates of P . aeruginosa .

J Neurosurg, 2000 Sep, 93(3), 477 - 9
Brain abscess related to metal fragments 47 years after head injury . Case report; Lee JH et al.; The authors report a case of symptomatic brain abscess in a 51-year-old man who presented with personality changes and generalized seizures . He had survived a grenade explosion injury during the Korean War 47 years previously . Computerized tomography scanning revealed multiple conglomerate rim-enhancing lesions and metallic foreign bodies in the right frontal lobe . The mass was totally removed and Pseudomonas aeruginosa was isolated from microbial cultures . Retained foreign bodies in the brain, whether bone or metal, should be removed at the time of injury if at all possible . If this cannot be accomplished, patients with such retained foreign bodies should be carefully monitored for life.

Infect Control Hosp Epidemiol, 2000 Aug, 21(8), 527 - 9
Epidemiology of nosocomial outbreaks: 14-year experience at a tertiary-care center; Ostrosky-Zeichner L et al.; Twelve nosocomial outbreaks over 14 years at a tertiary-care center in Mexico are described . Overall mortality was 25.8%, one half due to pneumonia . The most common organism was Pseudomonas aeruginosa . Incidence was three outbreaks per 10,000 discharges; outbreak-related infections comprised 1.56% of all nosocomial infections . Incidence in the intensive care unit was 10-fold higher.

Bioorg Med Chem, 2000 Jan, 8(1), 73 - 93
Antimicrobial effects of novel siderophores linked to beta-lactam antibiotics; Kline T et al.; As a strategy to increase the penetration of antibiotic drugs through the outer membrane of gram-negative pathogens, facilitated transport through siderophore receptors has been frequently exploited . Hydroxamic acids, catechols, or very close isosteres of catechols, which are mimics of naturally occurring siderophores, have been used successfully as covalently linked escorting moieties, but a much wider diversity of iron binding motifs exists . This observation, coupled to the relative lack of specificity of siderophore receptors, prompted us to initiate a program to identify novel, noncatechol siderophoric structures . We screened over 300 compounds for their ability to (1) support growth in low iron medium of a Pseudomonas aeruginosa siderophore biosynthesis deletion mutant, or (2) compete with a bactericidal siderophore-antibiotic conjugate for siderophore receptor access . From these assays we identified a set of small molecules that fulfilled one or both of these criteria . We then synthesized these compounds with functional groups suitable for attachment to both monobactam and cephalosporin core structures . Siderophore-beta-lactam conjugates then were tested against a panel of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus strains . Although several of the resultant chimeric compounds had antimicrobial activity approaching that of ceftazidime, and most compounds demonstrated very potent activity against their cellular targets, only a single compound was obtained that had enhanced, siderophore-mediated antibacterial activity . Results with tonB mutants frequently showed increased rather than decreased susceptibilities . suggesting that multiple factors influenced the intracellular concentration of the drugs.

Med J Malaysia, 1997 Mar, 52(1), 88 - 91
Severe combined immunodeficiency in a Malaysian child; Noh LM et al.; A 3-month-old Malay male infant presented with multiple infections (candidiasis, Pseudomonas aeruginosa, Cytomegalovirus), persistent pneumonia, intractable diarrhoea and failure to thrive . There was lymphopaenia affecting both T and B subsets . He developed Graft versus Host disease weeks following transfusion with non irradiated blood . In spite of aggressive microbicidal and supportive therapy including regular immunoglobulin infusions, the child succumbed to infection before a bone marrow transplant could be instituted.

Microbes Infect, 2000 Jul, 2(9), 1051 - 60
Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist; Lyczak JB et al.; Pseudomonas aeruginosa is an ubiquitous pathogen capable of infecting virtually all tissues . A large variety of virulence factors contribute to its importance in burn wounds, lung infection and eye infection . Prominent factors include pili, flagella, lipopolysaccharide, proteases, quorum sensing, exotoxin A and exoenzymes secreted by the type III secretion system.

Invest Ophthalmol Vis Sci, 2000 Sep, 41(10), 3019 - 25
Aging and PMN response to P . aeruginosa infection; Kernacki KA et al.; PURPOSE: Alterations in immune system function associated with aging may contribute to increased morbidity in this population of individuals . The current studies were performed to determine aging-related changes in polymorphonuclear neutrophil (PMN) function after corneal infection with Pseudomonas aeruginosa . METHODS: Total PMN number, macrophage inflammatory protein (MIP)-2 mRNA and protein expression, and ocular bacterial load were determined in 8-week- and 12-month-old inbred BALB/c mice at various times after infection with P . aeruginosa . In addition, 12-month-old mice were treated systemically with the MIP-2 polyclonal antibody (pAb) to determine the effects of MIP-2 neutralization on ocular disease and PMN recruitment . RESULTS: Histologically, PMN infiltration into the cornea of 12-month-old mice was delayed initially and was associated with an inability to reduce bacterial load at later postinfection (PI) times . In addition, a significantly greater number of PMNs were found in the cornea of 12-month-old mice at later PI times . The increase in PMN number in 12-month-old mice correlated with a persistence of MIP-2 expression in cornea at these later times . Systemic treatment of 12-month-old mice with neutralizing MIP-2 pAb versus normal rabbit serum (NRS) resulted in reduced corneal PMN number and ocular disease . CONCLUSIONS: These data provide evidence that persistence of PMN in the cornea of 12-month-old mice contributes to corneal tissue destruction after P . aeruginosa challenge . Further evidence also is provided that the chemoattractant MIP-2 contributes to the altered PMN response in these animals.

Crit Care Med, 2000 Aug, 28(8), 2737 - 41
Impact of quantitative invasive diagnostic techniques in the management and outcome of mechanically ventilated patients with suspected pneumonia; Sole Violan J et al.; OBJECTIVE: To assess how data obtained by invasive diagnostic techniques may affect management and outcome of patients with suspected ventilator-associated pneumonia (VAP), in comparison with noninvasive qualitative techniques . DESIGN: Prospective study . SETTING: An 18-bed medical and surgical intensive care unit . PATIENTS: A total of 91 patients suspected of having VAP were randomized into two groups . In group A (n = 45), quantitative cultures obtained by either bronchoscopic or nonbronchoscopic techniques were performed, whereas in group B (n = 43), patients were treated based on clinical judgment and nonquantitative tracheal aspirates cultures . Three patients were excluded because of the absence of follow-up . RESULTS: In patients with positive cultures, therapeutic changes were made in 20 patients . In four patients (three from group A and one from group B, p = NS), initial empirical antibiotic treatment was modified because the isolated microorganisms were not susceptible (all of them had late-onset pneumonia) . The isolated organisms responsible for antibiotic modifications were methicillin-resistant Staphylococcus aureus (three patients) and Pseudomonas aeruginosa (one patient) . In three patients, the antimicrobial therapy was considered inappropriate because the isolated microorganisms were multiresistant and treated with only one effective antibiotic . In 13 patients (ten from group A and three from group B, p < .05), treatment was changed to select a narrower spectrum antibiotic . No therapeutic modifications were made in patients with negative cultures based on the results of quantitative cultures . The overall mortality was 22.2% in group A and 20.9% in group B . There were no differences in intensive care unit stay or days of mechanical ventilation (23.67+/-3.15 vs . 22.42+/-3.01 and 19.99+/-2.88 vs . 19.24+/-3.04, respectively) . CONCLUSIONS: In our study population, the routine use of quantitative invasive diagnostic tools is not justified in the setting of ventilated patients clinically suspected of having nosocomial pneumonia.

Ophthalmologica, 2000 Sep-Oct, 214(5), 324 - 31
Contamination of contact-lens-related sources with Pseudomonas aeruginosa; Micallef C et al.; The studies conducted sought to estimate contamination of contact lens (CL) storage cases and commercial solutions with Pseudomonas aeruginosa as well as to investigate everyday sources, which the CL wearer uses or comes into contact with . The studies revealed that 13 . 1% of storage lens cases and 5.1% of commercial solutions in use were positive for the bacterium . Ten hermetically sealed CL solutions were tested for possible contamination, but these all proved to be sterile . In addition, environmental samples, of both a solid and liquid nature, yielded relatively high contamination rates with this opportunistic bacterium . Results showed that common sources, which the CL wearer is in contact with, possess P . aeruginosa and this may indeed predispose to corneal infections .

Ophthalmology, 2000 Sep, 107(9), 1661 - 4; discussion 1664-5
Primary implant placement with evisceration in patients with endophthalmitis; Dresner SC et al.; OBJECTIVE: To evaluate the efficacy of primary orbital implant placement with evisceration in patients with endophthalmitis and blind eyes . DESIGN: Retrospective noncomparative case series . PARTICIPANTS: Eleven patients with endophthalmitis and blind eyes underwent evisceration by two surgeons between 1994 and 1998 . INTERVENTION: Evisceration and primary orbital implant placement . MAIN OUTCOME MEASURES: All patients were evaluated for implant exposure and successful fitting of their prostheses . RESULTS: Ten of 11 patients had uneventful postoperative courses and successful prosthetic fitting . One patient with Pseudomonas aeruginosa endophthalmitis had an implant exposure successfully treated with a fascia lata patch . CONCLUSIONS: Primary orbital implant placement with evisceration in patients with endophthalmitis is an acceptable treatment, eliminating the need for open evisceration and subsequent delayed orbital implant placement.

Anal Biochem, 2000 Sep 10, 284(2), 388 - 93
Assay for lytic transglycosylases: a family of peptidoglycan lyases; Blackburn NT et al.; An assay has been developed to monitor the activity of the lytic transglycosylases which does not involve the use of radiolabel . Samples of lytic transglycosylase were incubated with isolated and purified insoluble peptidoglycan as substrate for varying lengths of time . Residual insoluble material was removed by ultracentrifugation in a microfuge and the solubilized components were treated with sodium borohydride prior to acid hydrolysis . The optimal conditions for this acid hydrolysis were established to be incubation at 96 degrees C for 1 h in 6 M HCl, in vacuo . The hydrolyzed samples were subjected to amino acid/sugar analysis by cation-exchange chromatography on a Beckman System Gold amino acid analyzer . To effect a clear resolution of muramic acid from serine and glutamic acid, the equilibration buffer was modified to be composed of 33 mM sodium citrate, pH 3.12 . The product of the lyase reaction of the lytic transglycosylases are 1,6-anhydromuramyl residues, which are not reduced by the sodium borohydride treatment . On the other hand, the muramyl residues arising at the reducing ends of peptidoglycan after treatment with muramidases (hydrolyases) are reduced to muramitol residues, which elute from the amino acid analyzer prior to aspartic acid . This assay thus distinguishes the activity of the two enzymes and was applied to determine the initial activities of increasing concentrations of a soluble derivative of lytic transglycosylase B from the opportunistic pathogen Pseudomonas aeruginosa .

Lung, 2000, 178(4), 235 - 48
Lipopolysaccharide tolerance in relation to intrabronchial influx of neutrophils in the rat; Shimada M et al.; Lipopolysaccharide (LPS) is a potent chemotactic component for polymorphonuclear leukocytes (PMN, neutrophils) . Since LPS tolerance was first described, many studies have been reported about the hyporesponsiveness in vitro corresponding to attenuating production of proinflammatory cytokines . We hypothesized that in vivo daily exposure to LPS stimuli impairs neutrophil accumulation in the rat airway . Interleukin 8 (IL-8) and/or CXC-chemokine, a neutrophil chemoattractant and activating cytokine, have been implicated as proinflammatory mediators in gram-negative respiratory tract infections . It is possible that the tolerance to LPS has occurred in relation to this chemoattractant cytokine production . To settle this issue, we examined whether the neutrophil count in bronchoalveolar lavage fluid (BALF) decreases after daily inhalation of Pseudomonas aeruginosa LPS into the rat airway . Repeated inhalation of LPS into the airway resulted in reduction in neutrophil recruitment . We measured rat CXC-chemokine (rat GRO/CINC1) levels in recovered BALF . There were noted reductions of rat GRO corresponding to the diminished neutrophil trafficking . We also confirmed that the HLA-DR positive lymphocyte number in BALF gradually increased after daily inhalation of LPS . These results suggest that continuous stimuli of LPS mitigate the accumulation of inflammatory cells in the airway by reducing chemokine production with a consequent change in the appearance of local inflammation to a chronic state.

J Bacteriol, 2000 Sep, 182(18), 5251 - 5
The amino terminus of Pseudomonas aeruginosa outer membrane protein OprF forms channels in lipid bilayer membranes: correlation with a three-dimensional model; Brinkman FS et al.; Pseudomonas aeruginosa OprF forms 0.36-nS channels and, rarely, 2- to 5-nS channels in lipid bilayer membranes . We show that a protein comprising only the N-terminal 162-amino-acid domain of OprF formed the smaller, but not the larger, channels in lipid bilayers . Circular dichroism spectroscopy indicated that this protein folds into a beta-sheet-rich structure, and three-dimensional comparative modeling revealed that it shares significant structural similarity with the amino terminus of the orthologous protein Escherichia coli OmpA, which has been shown to form a beta-barrel . OprF and OmpA share only 15% identity in this domain, yet these results support the utility of modeling such widely divergent beta-barrel domains in three dimensions in order to reveal similarities not readily apparent through primary sequence comparisons . The model is used to further hypothesize why porin activity differs for the N-terminal domains of OprF and OmpA.

J Biomol NMR, 2000 Jul, 17(3), 239 - 55
Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy; Campbell AP et al.; The backbone dynamics of a 15N-labeled recombinant PAK pilin peptide spanning residues 128-144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys128-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Se r-Lys144) were probed by measurements of 15N NMR relaxation . This PAK(128-144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P . aeruginosa infection . The 15N longitudinal (T1) and transverse (T2) relaxation rates and the steady-state heteronuclear {1H}-15N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25 degrees C) and at pH 4.5 and 7.2 . Relaxation data was analyzed using both the 'model-free' formalism {Lipari, G . and Szabo, A . (1982) J . Am . Chem . Soc., 104, 4546-4559 and 4559-4570} and the reduced spectral density mapping approach {Farrow, N.A., Szabo, A., Torchia, D.A . and Kay, L.E . (1995) J . Biomol . NMR, 6, 153-162} . The relaxation data, spectral densities and order parameters suggest that the type I and type II beta-turns spanning residues Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively, are the most ordered and structured regions of the peptide . The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P . aeruginosa strains.

J Microbiol Methods, 2000 Aug, 41(3), 259 - 65
Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples; Jimenez L et al.; PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination . Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger . Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h . A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E . coli, S . aureus, and P . aeruginosa . However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A . niger DNA sequences . Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h . Low levels of microbial contamination were detected in all raw materials and products using PCR assays . Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.

Eur J Med Res, 2000 Aug 18, 5(8), 356 - 9
Relations between the frequency of the DeltaF 508 mutation and the course of pulmonary disease in cystic fibrosis patients infected with Pseudomonas aeruginosa; Rosenecker J; The course of pulmonary disease in cystic fibrosis is variable . There are controversial data on the impact of the type of mutation on the cystic fibrosis transmembrane conductance regulator (CFTR) gene on the course of pulmonary disease in CF . Since selected mutations of the CFTR gene appear to be associated with relatively mild disease, this study addressed the question whether the course of pulmonary disease in CF patients colonized with P . aeruginosa is influenced by the frequency of the DeltaF 508 mutation . For this study, we assessed FVC and FEV1 in 127 CF patients who attended regularly the Munich CF Center over a mean period of 43 +/- 16 (SD) months . Of these 127 patients, 69 (54.3%) were homozygous for the DeltaF 508 mutation, 42 (33.1%) were compound heterozygous for the DeltaF 508 mutation, and 16 (12.6%) did not carry the DeltaF 508 mutation on either chromosome . In homozygotes 59 (85.5%) out of 69 CF-patients were colonized with P . aeruginosa as compared with 27 (64.3%) out of 42 in heterozygotes (p <0.05) . The mean age of onset of P . aeruginosa colonization was 11.0 years, and there was no difference between the three groups . The mean FVC and FEV1 values did not differ significantly between the three genotype groups when P . aeruginosa infection was disregarded . However, when only P . aeruginosa colonized patients were compared FVC and FEV1 values were lower in heterozygotes than in the other two groups both at the beginning and at the end of the study . These findings indicate that the course of pulmonary disease in CF patients is at least partially influenced by the frequency of the DeltaF 508 mutation.

Am J Physiol Lung Cell Mol Physiol, 2000 Sep, 279(3), L452 - 9
Regulation and function of CCSP during pulmonary Pseudomonas aeruginosa infection in vivo; Hayashida S et al.; Clara cell secretory protein (CCSP) is a 16-kDa homodimeric polypeptide secreted by respiratory epithelial cells in the conducting airways of the lung . To assess the role of CCSP in bacterial inflammation and to discern whether CCSP expression is influenced by bacterial infection, CCSP-deficient {(-/-)} gene-targeted mice and wild-type mice were given Pseudomonas aeruginosa intratracheally . Infiltration by polymorphonuclear cells was significantly increased in the lungs of CCSP(-/-) mice 6 and 24 h after the administration of the bacteria . The number of viable bacteria isolated from the lungs in CCSP(-/-) mice was decreased compared with that in wild-type mice . Concentrations of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha were modestly increased after 6 and 24 h, respectively, in CCSP(-/-) mice . The concentration of CCSP protein in lung homogenates decreased for 1-5 days after infection and recovered by 14 days after infection . Likewise, CCSP mRNA and immunostaining for CCSP markedly decreased in respiratory epithelial cells after infection . CCSP deficiency was associated with enhanced pulmonary inflammation and improved killing of bacteria after acute pulmonary infection with P . aeruginosa . The finding that Pseudomonas infection inhibited CCSP expression provides further support for the concept that CCSP plays a role in the modulation of pulmonary inflammation during infection and recovery.

J Med Chem, 2000 Aug 10, 43(16), 3085 - 92
Structure-function studies of polymyxin B nonapeptide: implications to sensitization of gram-negative bacteria; Tsubery H et al.; Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived by enzymatic processing from the naturally occurring peptide polymyxin B, is able to increase the permeability of the outer membrane of Gram-negative bacteria toward hydrophobic antibiotics probably by binding to the bacterial lipopolysaccharide (LPS) . We have synthesized 11 cyclic analogues of PMBN and evaluated their activities compared to that of PMBN . The synthetic peptides were much less potent than PMBN in their capacity to sensitize Escherichia coli and Klebsiella pneumoniae toward novobiocin and to displace dansyl-PMBN from Escherichia coli LPS . Moreover, unlike PMBN, none of the analogues were able to inhibit the growth of Pseudomonas aeruginosa . The structural-functional features of PMBN were characterized and identified with regard to the ring size, the distance between positive charges and peptide backbone, the chirality of the DPhe-Leu domain, and the nature of the charged groups . Apparently, the structure of PMBN is highly specific for efficient perturbation of the outer membrane of Gram-negative bacteria as well as for LPS binding . The present study further increases our understanding of the complex PMBN-LPS and may, potentially, enable the design of compounds having enhanced permeabilization potency of the Gram-negative outer membrane.

Rev Clin Esp, 2000 Jun, 200(6), 301 - 4
{Postsurgical meningitis caused by Pseudomonas aeruginosa: study of 15 cases and review of the literature}; Rodriguez Guardado A et al.; OBJECTIVE: Pseudomonas aeruginosa meningitis is a rare condition which is usually associated with pathology in the ORL field, neurosurgery or local neurologic manipulations . The characteristics, epidemiology, and course of this entity were determined . METHODS: Fifteen episodes of nosocomial postsurgical Pseudomonas aeruginosa meningitis occurred between 1989 and 1996 were retrospectively analyzed . RESULTS: A previous cranioencephalic trauma was recorded in 46.6% of patients . The portals of entry included: intraventricular catheter (IC) (12 cases), CSF fistula (2 cases), and craniotomy (1 case) . In five occasions (41.6%) the microorganism was also recovered from the intraventricular catheter . Once culture results were available, therapy with active drugs against Pseudomonas was instituted and in 7 occasions was accompanied by the removal of IC . Eight patients eventually cured and two patients relapsed . The absence of cure was significantly associated with non-removal of the IC (p < 0.01) . The infection resulted in death in 26.6% of patients . CONCLUSIONS: Postsurgical Pseudomonas aeruginosa meningitis is an entity of growing relevance . It is associated with relevant morbi-mortality . Catheter removal is essential to obtain a favorable outcome.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2242 - 6
Contribution of the MexX-MexY-oprM efflux system to intrinsic resistance in Pseudomonas aeruginosa; Masuda N et al.; To test the possibility that MexX-MexY, a new set of efflux system components, is associated with OprM and contributes to intrinsic resistance in Pseudomonas aeruginosa, we constructed a series of isogenic mutants lacking mexXY and/or mexAB and/or oprM from a laboratory strain PAO1, and examined their susceptibilities to ofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin . Loss of either MexXY or OprM from the MexAB-deficient mutant increased susceptibility to all agents tested, whereas loss of MexXY from the MexAB-OprM-deficient mutant caused no change in susceptibility . Introduction of an OprM expression plasmid decreased the susceptibility of the mexAB-oprM-deficient-/mexXY-maintaining mutant, yet caused no change in the susceptibility of a mexAB-oprM- and mexXY-deficient double mutant . Immunoblot analysis using anti-MexX polyclonal rabbit serum generated against synthetic oligopeptides detected expression of MexX in the PAO1 cells grown in medium containing tetracycline, erythromycin, or gentamicin, although expression of MexX was undetectable in the cells incubated in medium without any agent . These results suggest that MexXY induced by these agents is functionally associated with spontaneously expressed OprM and contributes to the intrinsic resistance to these agents.

Appl Microbiol Biotechnol, 2000 Jul, 54(1), 37 - 43
In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa; Qi Q et al.; For the first time, the purification has been achieved of the type II polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa applying N-terminal His6-tag fusions and metal chelate affinity chromatography . In vivo His6-tagged PHA synthase activity was confirmed by functional expression of the corresponding genes in Escherichia coli, and PHA synthase activity could also be measured in vitro with the enzymes . The specific enzyme activity of PHA synthases PhaC1 and PhaC2 was 0.039 U mg(-1) and 0.035 U mg(-1) protein, respectively . Kinetic studies showed a lag phase for both PHA synthases using (R,S)-3-hydroxydecanoyl-CoA as substrate . Specific enzyme activity was increased to 0.055 U mg(-1) when the phasin GA24 from Ralstonia eutropha was added to the assay . CoA inhibited PHA synthase activity, and a Ki of 85 microM was determined . A two-enzyme system was established, employing commercially available acyl-CoA synthetase and PHA synthase, which allowed the in vitro de novo PHA granule formation and the in vitro synthesis of poly(3-hydroxydecanoate) exhibiting a weight average molar mass of 9.8 x 10(4) g mol(-1), and which occurred independently of pre-existing PHA granules.

Thorax, 2000 Sep, 55(9), 795 - 7
Increased sputum amino acid concentrations and auxotrophy of Pseudomonas aeruginosa in severe cystic fibrosis lung disease; Thomas SR et al.; BACKGROUND: Pseudomonas aeruginosa may undergo a phenotypic change from the wild (prototrophic) type to an auxotrophic phenotype in the course of respiratory infection in patients with cystic fibrosis . The clinical significance of this is unclear . A study was undertaken to investigate whether the presence of auxotrophs of P aeruginosa in the sputum of patients with cystic fibrosis correlated with severity of respiratory disease, and whether increased sputum concentrations of amino acids were associated with the emergence of these forms . METHODS: Sixty adult patients with cystic fibrosis, colonised by P aeruginosa, were recruited and baseline clinical data including lung function were recorded . Serial sputum samples were obtained before, during, and after infective exacerbations where possible . These samples were used for routine microbiological culture, assessment of auxotrophy of P aeruginosa, measurement of amino acid content, and neutrophil elastase assay . RESULTS: Auxotrophy was common in patients with cystic fibrosis and 20 (33%) had a mean percentage auxotroph count of more than 50% total cfu/ml . The mean percentage auxotroph count was inversely correlated with forced expiratory volume in one second (FEV(1); tau = -0.194, p = 0.031) . The median sputum amino acid concentration of the group was 12.5 mmol/l (range 0.13-40.6) . The mean amino acid concentration in 33 subjects during infective exacerbations was 18.2 mmol/l (95% CI 15.1 to 21.3) compared with 12.3 mmol/l (95% CI 9.8 to 14.8) when well (p = 0.001) . The amino acid content of sputum was inversely correlated with FEV(1) (tau = -0.253, p = 0.005) . CONCLUSIONS: P aeruginosa frequently exhibits auxotrophy in patients with cystic fibrosis, particularly in those with severe underlying pulmonary disease . The sputum amino acid content of patients with cystic fibrosis is high during infective exacerbations and correlates with pulmonary disease severity.

Pathol Biol (Paris), 2000 Jun, 48(5), 472 - 7
{Surveillance of Pseudomonas aeruginosa sensitivity to antibiotics in France and distribution of beta-lactam resistance mechanisms: 1998 GERPB study}; Cavallo JD et al.; In a prospective study carried out during a three-week period in October 1998 in 13 teaching hospitals, 735 non-repetitive isolates of Pseudomonas aeruginosa were collected . In patients presenting cystic fibrosis (70 strains), the main serotypes isolated were O:6 (14.3%) and O:1 (14.3%) . Serotypes O:11 and O:12 were exceptional . In other patients (665 strains), the most frequent serotypes were O:6 (15.9%), O:11 (15.6%), O:1 (10.7%) and O:12 (9.2%) . The antibiotic susceptibility rates were as follows (respectively, non-cystic fibrosis and cystic fibrosis strains): ticarcillin, 55 and 59%, piperacillin, 71 and 67%, ceftazidime, 75 and 67%, cefepime, 56 and 43%, cefpirome, 37 and 21%, aztreonam, 57 and 56%, imipenem, 83 and 70%, amikacin, 69 and 33%, ciprofloxacin, 56 and 61% and fosfomycin, 33 and 43% . Serotype O:12 was the least susceptible to antibiotics . Forty-five percent of the non-cystic fibrosis strains presented intermediate susceptibility or resistance to ticarcillin . The most frequent mechanisms of resistance were: non-enzymatic resistance (14.3%), overproduction of the constitutive cephalosporinase (13.8%), production of transferable beta-lactamase (8.6%) and a combination of these mechanisms (4.2%) . Among cystic fibrosis strains, resistance to beta-lactam antibiotics was mainly due to overproduction of the constitutive cephalosporinase (18.6%), whereas production of a transferable beta-lactamase was rare (1.4%) . Susceptibility to aminoglycosides and fluoroquinolones was less frequent in isolates producing transferable beta-lactamases and/or overproducing cephalosporinase . Decreased susceptibility to imipenem was more frequent in strains presenting a high level of cephalosporinase production . Among the cephalosporins, cefepime was the least affected by the overproduction of constitutive cephalosporinase . Ceftazidime remained the most efficient antibiotic against both susceptible isolates and strains presenting a non-enzymatic or PSE-1 penicillinase-producing mechanism.

Klin Monatsbl Augenheilkd, 2000 Jul, 217(1), 37 - 42
{Polymerase chain reaction (PCR) for microbiological diagnosis in refractory infectious keratitis: a clinical study in 16 patients}; Lohmann CP et al.; BACKGROUND: The identification of the causative pathogen in infectious keratitis is possible in only 60% of the cases . The aim of this study was to show if this number increases by the use of PCR . PATIENTS AND METHODS: In a series of 16 eyes with infectious keratitis corneal specimens were collected for culture and PCR . Serology (HSV, VZV, and Borrelia) was performed in all eyes, with exception of the 4 eyes presenting an acute form of keratitis, which obviously was bacterial origin . RESULTS: In all 4 cases of acute keratitis the causative pathogen (Pseudomonas aeruginosa) was detected by both culture and PCR . Of the remaining 12 eyes PCR was capable to identify the causative pathogen in 11 eyes . In 3 eyes herpes simplex virus was detected, in 3 eyes Moraxella catharalis, in 2 eyes Borrelia burgdorferii, in 2 eyes varizella zoster virus, and in 1 eye Bartonella henselae . Culture was positive in only 2 eyes, infected by Moraxella catharalis . CONCLUSIONS: PCR is a useful supplement in the microbiological diagnostic of infectious keratitis, in particular if only a small amount of pathogens are available (non-acute form) or if the eye has been treated by antibiotics prior to the microbiological diagnostic.

Infect Immun, 2000 Sep, 68(9), 4850 - 5
Characterization of a shiga toxin 2e-converting bacteriophage from an Escherichia coli strain of human origin; Muniesa M et al.; An infectious Shiga toxin (Stx) 2e-converting bacteriophage (phiP27) was isolated from Stx2e-producing Escherichia coli ONT:H(-) isolate 2771/97 originating from a patient with diarrhea . The phage could be transduced to E . coli laboratory strain DH5alpha, and we could show that lysogens were able to produce biologically active toxin in a recA-dependent manner . By DNA sequence analysis of a 6,388-bp HindIII restriction fragment of phiP27, we demonstrated that the stx(2e) gene was located directly downstream of ileZ and argO tRNA genes . Although no analogue of an antiterminator Q encoding gene was present on this fragment, a lysis cassette comprising two holin genes which are related to the holin genes of Pseudomonas aeruginosa phage phiCTX and a gene homologous to the endolysin gene gp19 of phage PS3 were detected . The results of our study demonstrated for the first time that Stx2e can be encoded in the genome of an infectious bacteriophage.

Wiad Lek, 2000, 53(3-4), 214 - 8
{Malignant external otitis: a rare complication after autologous bone marrow transplantation}; Krawczyk-Kulis M et al.; We present a case of 39 year old woman who developed malignant external otitis (m.e.o.) of Pseudomonas aeruginosa aetiology during pancytopenia after autologous bone marrow transplantation (ABMT) . The infection was probably of endogenous origin . 7 days before ABMT otolarygological examination including otoscopy and external ear lavage was performed . Slight inflammatory reaction of external ear was accompanied by the massive involvement of middle ear followed by infiltration of petrous pyramid and mastoid process and finally facial and vestibulocochlear nerve paralysis . Initially the symptoms indicated subarachnoid haemorrhage . Mononuclear cells detected in cerebrospinal fluid as well as CT scan were suggestive of leukaemic infiltration . The latter was negated by immunophenotyping of cerebrospinal fluid cells and MR imaging . Antibiotic therapy resulted in clinical improvement . Life-threatening complications are not frequent after ABMT (transplant related mortality--14/310 (4.5%) in our center) . We have met m.e.o . for the first time . At present--13 months after ABMT the patient shows slight symptoms of nerve VII and VIII paresis and remains in complete remission of acute leukaemia . We emphasize the importance of proper preparation of patients for high dose chemotherapy followed by bone marrow transplantation as well as diagnostic difficulties related to pancytopenia.

J Aerosol Med, 2000 Spring, 13(1), 11 - 6
Basis for nebulized antibiotics: droplet characterization and in vitro antimicrobial activity versus Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; Todisco T et al.; The aims of this study were to (1) quantify the particle size characteristics of several antibiotics considered suitable for aerosol therapy after aerosolization with the PARI IS/2 nebulizer (Pari GmbH, Sarnberg, Germany) and (2) determine the degree to which in vitro antimicrobial activity of these antibiotics is maintained after nebulization . The aerosolized drugs were tobramycin sulfate, streptomycin, and imipenem, with saline solution as the control . Mean mass aerodynamic diameter of the nebulized drugs was 3.25 microns for tobramycin, 2.26 microns for imipenem, and 2.38 microns for streptomycin . In vitro tests showed that tobramycin and imipenem were unaltered in their bacteriostatic activity against strains of Escherichia coli (American Type Culture Collection {ATCC} 25922) and Staphylococcus aureus (ATCC 29213) as well as against Pseudomonas aeruginosa (ATCC 27853) with minimal inhibitory concentration (MIC) values less than 0.3 microgram/mL . Nebulized streptomycin showed significantly higher MIC values against P . aeruginosa (ATCC 27853) . These results suggest that tobramycin and imipenem may be prescribed as an aerosol generated by jet nebulization (PARI IS/2) to treat S . aureus, E . coli, and P . aeruginosa infections without any risk of altering the drugs minimum bacteriostatic activity by the nebulization process . Aerosolization of streptomycin with this nebulizer may not be as effective against P . aeruginosa because it seems to alter the bacteriostatic activity.

J Appl Microbiol, 2000 Jul, 89(1), 158 - 68
Role of rhamnolipid biosurfactants in the uptake and mineralization of hexadecane in Pseudomonas aeruginosa; Beal R et al.; A study was undertaken to investigate the mechanisms for biosurfactant-enhanced hexadecane uptake into Pseudomonas aeruginosa . Two strains of Ps . aeruginosa were studied, one producing rhamnolipids (PG201) and the other rhamnolipid deficient (UO299) . Rhamnolipids produced by PG201 acted to increase the solubility of n-hexadecane in the culture medium (from 1.84 to 22.76 microg l(-1) . Rates of(l4)C-n-hexadecane uptake and mineralization were higher in PG201 than in UO299 . However, the degree of difference was lower than expected . Additional studies were carried out on the cell surface properties of the two strains . During growth on n-hexadecane, the cell surface hydrophobicity of both PG201 (50.5%) and UO299 (33.7%) increased compared with that observed in water-soluble growth substrates (7-8%) . Studies were also carried out to ascertain any energy requirements for the transport of n-hexadecane into Ps . aeruginosa cells . The addition of CCCP (an inhibitor of cytochrome oxidase which thereby blocks oxidative phosphorylation) at a range of concentrations caused a marked decrease in n-hexadecane uptake, indicating that n-hexadecane uptake in Ps . aeruginosa is an energy-dependent process . These studies support the hypothesis of alkane transport into microbial cells by direct contact with larger alkane droplets and by pseudosolubilization . Also, it appears that both mechanisms occur simultaneously.

J Appl Microbiol, 2000 Jul, 89(1), 32 - 9
Microemulsions are membrane-active, antimicrobial, self-preserving systems; Al-Adham IS et al.; Microemulsions are physically stable oil/water systems that have potential use as delivery systems for many pharmaceuticals which are normally of limited use due to their hydrophobicity, toxicity or inability to access the site of action . It has been suggested that microemulsions are self-preserving antimicrobials in their own right, although there is little evidence to support this . In this experiment, microemulsions of various compositions were formulated and tested for their stability and antimicrobial action . The physical stability of the different microemulsions was assessed by centrifugation at 4000g and by storage in a water bath at 37 degrees C for one month, during which no phase separation was observed . The antimicrobial activity of the microemulsions was tested using the compendial method, observation of the kinetics of killing, and transmission electron microscopy (TEM) of microemulsion-exposed cultures of Pseudomonas aeruginosa PA01 . These latter experiments on Ps . aeruginosa indicated distinct signs of membrane disruption . The results indicated that the microemulsions are self-preserved, and that their killing of microbial cultures is very rapid and may be the result of membrane activity.

J Mol Microbiol Biotechnol, 1999 Nov, 1(2), 289 - 93
Novel phosphotransferase systems revealed by bacterial genome analysis: the complete repertoire of pts genes in Pseudomonas aeruginosa; Reizer J et al.; We herein describe all genes encoding constituents of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in the 6Mbp genome of the opportunistic human pathogen, Pseudomonas aeruginosa . Only four gene clusters were found to encode identifiable PTS homologues . These genes clusters encode novel multidomain proteins, two complete sugar-specific PTS phosphoryl transfer chains for the metabolism of fructose and N-acetylglucosamine, and a complex regulatory system that may function to coordinate carbon and nitrogen metabolism . No previously characterized organism has been shown to exhibit such a novel and restricted complement of PTS proteins.

Gene, 2000 Aug 8, 253(2), 323 - 30
Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa; Adewoye LO et al.; The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein . We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene . The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins . The genomic copy of the upstream ORF was mutagenized by homologous recombination . Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in {14C} glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter . It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system . Multiple alignment analysis revealed that the P . aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

New Microbiol, 2000 Jul, 23(3), 319 - 27
Molecular epidemiology of Pseudomonas aeruginosa from cystic fibrosis in Sicily: genome macrorestriction analysis and rapid PCR-ribotyping; Agodi A et al.; This study addresses the epidemiologic relatedness of a collection of Pseudomonas aeruginosa isolates from cystic fibrosis patients attending the Pediatric Clinic, Catania, Sicily . Genome macrorestriction analysis after pulsed field gel electrophoresis (PFGE) was used to characterise all strains . Furthermore, a rapid typing procedure, developed in this study, based on polymerase chain reaction amplified ribosomal DNA spacer polymorphisms (PCR-ribotyping), straight from bacterial cultures, was used . On the basis of macrorestriction analysis after PFGE, persistence of infection was shown in all patients; two cross-transmission episodes were identified in the nosocomial as well as in the familiar environment . PCR-ribotyping proved to be useful for a DNA-based identification test, suitable for screening purposes . The rapid amplification protocol here tested is proposed to evaluate the discriminatory power of other specific target sequences in PCR-based typing assays, for epidemiologic purposes.

APMIS, 2000 May, 108(5), 329 - 35
The immune response to chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is predominantly of the Th2 type; Moser C et al.; Most cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa lung infection have a persistent acute type lung inflammation dominated by polymorphonuclear neutrophils (PMN) and a pronounced antibody response against P . aeruginosa . We speculated whether this immune response in CF is of the Th2 type and whether a change to a Th1 type immune response could improve the prognosis . Therefore, we studied 14 CF patients with (CF +P) and 14 CF patients without (CF -P) chronic P . aeruginosa lung infection . The specific production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by peripheral blood mononuclear cells was determined . Cells from CF +P patients had lower IFN-gamma (p<0.05) and higher IL-4 (p<0.005) production as compared to cells from CF -P patients . Furthermore, a positive correlation between IFN-gamma production and lung function was found (FVC: Rho = 0.637; p<0.03; FEV1: Rho=0.524; p<0.07) . We conclude that a Th2 type immune response is most frequent in CF patients with chronic P . aeruginosa lung infection, and the patients with a Th1-dominated immune response had the best lung function . The clinical implication is that a change to a Th1 type immune response might improve the prognosis in these patients.

Metab Eng, 2000 Apr, 2(2), 92 - 103
Pm promoter expression mutants and their use in broad-host-range RK2 plasmid vectors; Winther-Larsen HC et al.; By coupling the Pm/xylS promoter system to minimal replicons of the broad-host-range plasmid RK2 we recently showed that such vectors are useful for both high- and low-level inducible expression of cloned genes in gram-negative bacteria . In this report, we extend this potential by identifying point mutations in or near the -10 transcriptional region of Pm . Point mutations leading to gene-independent enhancements of expression levels of the induced state or reduced background expression levels were identified using Escherichia coli as a host . By combining these mutations an additive effect in expression levels from the constructed Pm was observed . The highest induced expression level was obtained by inserting an E . coli consensus sigma70 - 10 recognition region . Most of the remaining activities in the reduced-background mutations appeared to originate from a transcriptional start site other than Pm . The effects of some of these mutations were also analyzed in Pseudomonas aeruginosa and were found to act similarly, but less pronounced in this host.

Paediatr Drugs, 1999 Oct-Dec, 1(4), 283 - 9
Treatment of otitis externa in children; Brook I; Inflammation of the external auditory canal can be localised or diffuse, and acute or chronic . Predisposing conditions include external trauma, loss of the canal's protective coating, maceration of the skin from water or humidity, and glandular obstruction . Acute otitis externa is generally caused by Pseudomonas aeruginosa or Staphylococcus aureus . Management of patients with otitis externa includes debridement, topical therapy with acidifying and antimicrobial agents, and systemic antimicrobial therapy when indicated . The management of patients with chronic otitis externa includes cleansing and debridement accompanied by topical acidifying and drying agents . This is followed by topical antibiotics and corticosteroid preparations . Surgery is mainly used to allow cleansing and aeration and/or removal of the scarred tissue . Patients with acute localised otitis externa (furunculosis) are treated with local heat and systemic antibiotics in the inflammatory stage, and drainage in the abscess state . Mycotic external otitis is managed with topical acidifying and antifungal agents, while viral (herpes) infection is treated with topical and systemic aciclovir (acyclovir) . Patients with necrotising (malignant) external otitis, which is mainly caused by P . aeruginosa and S . aureus, are treated with systemic antibiotics and, rarely, by surgical debridement . Therapy for eczematous otitis externa is first directed at the secondary infection, and thereafter at the primary dermatological condition . Prevention of recurrent external otitis is aimed at minimising ear canal trauma and the avoidance of exposure to water . Preventative use of topical acidifying agents or 70% alcohol is also advocated.

Am J Respir Crit Care Med, 2000 Aug, 162(2 Pt 1), 481 - 5
Tobramycin solution for inhalation reduces sputum Pseudomonas aeruginosa density in bronchiectasis; Barker AF et al.; We conducted a placebo-controlled, double-blind, randomized study to evaluate the microbiological efficacy and safety of inhaled tobramycin for treatment of patients with bronchiectasis and Pseudomonas aeruginosa . Patients were randomly assigned to receive either tobramycin solution for inhalation (TSI) (n = 37) or placebo (n = 37), which was self-administered twice daily for 4 wk and followed by 2-wk off-drug . At Week 4, the TSI group had a mean decrease in P . aeruginosa density of 4.54 log(10) colony-forming units (cfu)/g sputum compared with no change in the placebo group (p < 0.01) . At Week 6, P . aeruginosa was eradicated in 35% of TSI patients but was detected in all placebo patients . Investigators indicated that 62% of TSI patients showed an improved medical condition compared with 38% of placebo patients (odds ratio = 2.7, 95% confidence interval {CI} 1.1 to 6.9) . Tobramycin-resistant P . aeruginosa strains developed in 11% of TSI patients and 3% of placebo patients (p = 0.36) . The mean percent change in FEV(1) percent predicted from Week 0 to Week 4 was similar for the TSI and placebo groups (p = 0.41) . More TSI-treated patients than placebo patients reported increased cough, dyspnea, wheezing, and noncardiac chest pain, but the symptoms did not limit therapy . Additional study is warranted to further evaluate TSI in bronchiectasis patients.

Anal Biochem, 2000 Aug 15, 284(1), 29 - 34
Noninvasive tracing of recombinant proteins with "fluorophenylalanine-fingers"; Minks C et al.; High-level residue-specific replacement of phenylalanine residues in recombinant human annexin V and azurin from Pseudomonas aeruginosa with o-fluorophenylalanine, m-fluorophenylalanine, and p-fluorophenylalanine has been achieved using the selective pressure incorporation method . Incorporation was confirmed analytically and by UV spectroscopy while the secondary and tertiary structures of these protein mutants in solution remained unchanged upon the effected substitutions . Fluorinated phenylalanines alone and when integrated into proteins exhibit two characteristic and prominent shoulders ("fingers") in the UV spectrum in the range of 260-270 nm, which do not overlap with the contributions of tyrosine and tryptophan residues in the protein UV spectra . Thus, the presence of such "fluorophenylalanine fingers" ("FF fingers") opens a new spectral window to identify the labeled target protein among other nonlabeled cellular proteins in preparative work by simple UV spectroscopy . In the coming era of proteomics such a reliable, cheap, and easy reproducible methodology might have a great potential for speeding up the identification and characterization of target molecules in the total protein output from the genomes of a variety of organisms .

J Med Microbiol, 2000 Aug, 49(8), 701 - 7
Prolonged survival of mice with Pseudomonas aeruginosa-induced sepsis by rIL-12 modulation of IL-10 and interferon-gamma; Yamaguchi T et al.; Interleukin-12 (IL-12) is thought to play an important role as a modulator of levels of IL-10 and interferon-gamma (IFN-gamma) . To address the therapeutic effects of rIL-12 in an endogenous sepsis model in mice, which closely mimics the pathophysiology of septicaemia in man, the effects of rIL-12 on the levels of cytokines such as IL-10 and IFN-gamma, and on the survival of septic mice infected with Pseudomonas aeruginosa PAO1 were examined . First, in the endogenous sepsis model, the serum levels of IFN-gamma and IL-10 remained normal until days 8 and 10, respectively, when significant rises were seen . On day 11, levels of IFN-gamma returned to normal, but levels of IL-10 remained high . Interestingly, the IL-10 serum level reached a maximum 2 days later than the IFN-gamma serum level . In the light of these results, septic mice were given 0.01 microg of rIL-12 by intraperitoneal injection and the serum levels of endogenous cytokines and the survival times were examined . Mice treated with rIL-12 on days 5, 6 and 7 after infection survived significantly longer than control septic mice treated with saline only . Treatment with rIL-12 also led to a significant increase of the serum IFN-gamma level and a decrease of the serum IL-10 level on day 11 . These results suggest that rIL-12 exerts therapeutic activity against endogenous sepsis caused by P . aeruginosa by stimulating proinflammatory responses and attenuating anti-inflammatory responses.

Eur Respir J, 2000 Jul, 16(1), 146 - 9
Endothelin-1 in stable bronchiectasis; Zheng L et al.; Endothelin (ET)-1 has been suggested to promote neutrophil adhesion to endothelium, migration to inflamed areas, and release of elastase . ET-1 might therefore play a role in the pathogenesis of bronchiectasis, a chronic inflammatory and infective airway disease which is still poorly understood . Thirty five patients with stable bronchiectasis (20 females, mean age+/-SD 49.1+/-15.0 yrs) and 18 control subjects (8 females, 49.4+/-11.3 yrs) were recruited prospectively . The ET-1 levels in serum and sputum were measured by commercially available enzyme linked immunosorbent assay (ELISA) kits . Patients with Pseudomonas aeruginosa in their sputum had a significantly higher serum level of ET-1 (median 25.8, interquartile range 13-43.9 pg x mL(-1)) than patients without P . aeruginosa (0, 0-10.5 pg x mL(-1); p=0.0004) and healthy control subjects (4.6, 0-16.3 pg x mL(-1); p=0.002) . However, patients with and without P . aeruginosa infection had no significant difference in sputum ET-1 level (p=0.15) . There was no correlation between serum or sputum ET-1 levels with the serum and sputum levels of the interleukin (IL)-1beta, IL-8 and tumour necrosis factor (TNF)-alpha; the number of bronchiectasis lung lobes; and spirometry . Serum ET-1 level correlated with 24 h sputum volume for the bronchiectasis patients (r=0.51, p=0.002) . The results, therefore, suggest a significant pathogenic role for endothelin-1 among Pseudomonas aeruginosa-infected patients with bronchiectasis . Further studies should be performed to evaluate the clinico-pathological correlation and expression of endothelin-1 in bronchiectasis.

Masui, 2000 Jul, 49(7), 724 - 31
{Pretreatment of acid-induced lung injury with specific neutrophil elastase inhibitor ONO-5046 did not exasperate the subsequent bacterial lung infection by Pseudomonas aeruginosa}; Kudoh I et al.; Patients with acid lung injuries are at high risk for bacterial pulmonary infections which commonly occur several days after the acid aspiration . We reported that a specific neutrophil elastase inhibitor ONO-5046 inhibited the multi-organ injury caused by acid-instillation into the lung . In this study, we evaluated the effect of ONO-5046 on lung infection by Pseudomonas aeruginosa (PAO-1:Ps.) following acid-induced lung injury in rat lungs . Animals received 0.2 ml of hydrochloric acid (pH = 1) into the right lungs . Pretreated animals were administered ONO-5046 (30 mg.kg-1) i.v . 15 min . before acid instillation . Other groups received vehicle (saline) . Twenty four hours later, they were instilled with 0.1 ml of Ps . 1 x 10(8) cfu into the left lungs . Four hours after bacterial challenge, the animals were deeply anesthetized and killed . Bronchoalveolar lavage was done on each lung separately to evaluate neutrophil elastase activity, neutrophil number and protein permeability of lung endothelium and epithelium . The numbers of Ps . in the lungs were measured . In the Ps.-instilled lung, the number of Ps . or the protein permeability was not increased with ONO-5046 pretreatment compared with those in the untreated group . Pretreatment inhibited the exasperation of the protein permeability indirectly caused by Ps . infection in the acid-instilled lung . It was indicated that ONO-5046 could inhibit the indirect lung injury caused by acid-instillation into the lung without aggravating the subsequent bacterial infection.

Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9659 - 64
Interaction of pseudomonas aeruginosa with epithelial cells: identification of differentially regulated genes by expression microarray analysis of human cDNAs; Ichikawa JK et al.; Pseudomonas aeruginosa is an opportunistic pathogen that plays a major role in lung function deterioration in cystic fibrosis patients . To identify critical host responses during infection, we have used high-density DNA microarrays, consisting of 1,506 human cDNA clones, to monitor gene expression in the A549 lung pneumocyte cell line during exposure to P . aeruginosa . We have identified host genes that are differentially expressed upon infection, several of which require interaction with P . aeruginosa and the expression of the major subunit of type IV pili, PilA . Differential expression of genes involved in various cellular functions was identified, and we selected the gene encoding the transcription factor interferon regulatory factor 1 (IRF-1) for further analysis . The levels of the IRF-1 transcript increased 3- to 4-fold in A549 cells after adherence by P . aeruginosa . A similar increase of IRF-1 mRNA was observed in A549 cells exposed to wild-type P . aeruginosa when compared to an isogenic, nonpiliated strain . However, this difference was abolished when serum was present during the incubation of bacteria . Exposure of A549 cells to purified P . aeruginosa lipopolysaccharide did not result in a significant increase in IRF-1 mRNA . Although the P . aeruginosa-induced increased IRF-1 expression depends on the presence of bacterial adhesin, our findings do not preclude the possibility that other bacterial products are responsible for IRF-1 activation, which is enhanced by bacterial adherence to cells . These data show that microarray technology can be an important tool for studying the complex interplay between bacterial pathogens and host.

Microbiology, 2000 Aug, 146 ( Pt 8), 1891 - 9
Pseudomonas aeruginosa cystic fibrosis clinical isolates produce exotoxin A with altered ADP-ribosyltransferase activity and cytotoxicity; Gallant CV et al.; The role of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in the lung infections of cystic fibrosis (CF) patients is not well understood . Transcript-accumulation studies of bacterial populations in sputum reveal high levels of transcription of toxA, which encodes ETA, in some patients with CF . However, in general, tissue damage in the lungs of patients with CF does not seem to be consistent with a high level of expression of active ETA . To address this discrepancy the authors analysed the production and activity of ETA produced by a number of P . aeruginosa CF isolates . One CF isolate, strain 4384, transcribed toxA at levels similar to the hypertoxigenic strain PA103 but produced an ETA with reduced ADP-ribosyltransferase (ADPRT) activity . Complementation in trans of strain 4384 with the wild-type toxA and a mixed toxin experiment suggested the absence of inhibitory accessory factors within this strain . The toxA gene from strain 4384 was cloned and sequenced, revealing only three mutations in the gene, all within the enzymic domain . The first mutation changed Ser-410 to Asn . The second mutation was located within an alpha-helix, altering Ala-476 to Glu . The third mutation, Ser-515 to Gly, was found at the protein surface . To date, Ser-410, Ala-476 and Ser-515 have not been reported to play a role in the ADPRT activity of ETA . However, it may be the combination of these mutations that reduces the enzymic activity of ETA produced by strain 4384 . Expression of 4384 toxA and wild-type toxA in an isogenic strain revealed that 4384 ETA had 10-fold less ADPRT activity than wild-type ETA . ETA purified from strain 4384 also demonstrated 10-fold less ADPRT activity as compared to wild-type ETA . Cytotoxicity assays of purified ETA from strain 4384 indicated that the cytotoxicity of 4384 ETA is not reduced; it may be slightly more toxic than wild-type ETA . Analysis of five other CF isolates revealed a similar reduction in ADPRT activity to that seen in strain 4384 . Sequence analysis of the enzymic domain of toxA from the five CF strains identified a number of mutations that could account for the reduction in ADPRT activity . These results suggest that some CF isolates produce an ETA with reduced enzymic activity and this may partially explain the pathogenesis of chronic lung infections of CF due to P . aeruginosa.

J Biol Chem, 2000 Oct 27, 275(43), 33252 - 9
WbpO, a UDP-N-acetyl-D-galactosamine dehydrogenase from Pseudomonas aeruginosa serotype O6; Zhao X et al.; WbpO is associated with B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O6 . This protein is thought to catalyze the enzymatic conversion of UDP-N-acetyl-d-galactosamine (UDP-GalNAc) to UDP-N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA) . WbpO was overexpressed with a C-terminal hexahistidine tag . The soluble form of expressed WbpO (WbpO(Sol)) exhibited a secondary structure with 29.2% alpha-helix and 20.1% beta-strand . However, no enzymatic activity could be detected using either high performance anion exchange chromatography or capillary electrophoresis-mass spectrometry analysis . An insoluble form of expressed WbpO was purified in the presence of guanidine hydrochloride by immobilized metal ion affinity chromatography . After refolding, this preparation of WbpO (designated as WbpO(Rf)) exhibited stable secondary structure at pH 7.5 to 8.2, and it was enzymatically active . Capillary electrophoresis-mass spectrometry and tandem mass spectrometry analysis showed that WbpO(Rf) catalyzed the conversion of UDP-GalNAc to UDP-GalNAcA . 26 and 22% of the substrate could be converted to UDP-GalNAcA in the presence of NAD(+) and NADP(+) as the cofactors, respectively . The K(m) values of WbpO(Rf) for UDP-GalNAc, NAD(+), and NADP(+) were 7.79, 0.65, and 0.44 mm, respectively . WbpO(Rf) can also catalyze the conversion of UDP-GlcNAc to UDP-GlcNAcA . In conclusion, this is the first report of the overexpression, purification, and biochemical characterization of an NAD(+)/NADP(+)-dependent UDP-GalNAc dehydrogenase . Our results also complete the biosynthetic pathway for GalNAcA that is part of the O-antigen of P . aeruginosa serotype O6 lipopolysaccharide.

Mol Microbiol, 2000 Aug, 37(3), 561 - 73
Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1; Swanson BL et al.; The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene . We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1) . In this study, we searched the P . aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P . aeruginosa chromosome . Another PtxS operator site (OP2) was located 47 bp downstream of ptxS . DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2 . The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively . The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments . Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase . The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif . Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists . An isogenic mutant defective in ORF1 was constructed in the P . aeruginosa strain PAO1 . In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source . Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source . Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P . aeruginosa 2-ketogluconate-negative mutant . In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited . These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P . aeruginosa . Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively . (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.

Mol Microbiol, 2000 Jul, 37(2), 287 - 99
Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells; Pederson KJ et al.; ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways . The N-terminus (residues 1-234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232-453) encodes an ADP-ribosyltransferase . Utilizing a series of N-terminal deletion peptides of ExoS and an epitope-tagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity . N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS . The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90-234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization . ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol . Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.

Clin Exp Immunol, 2000 Aug, 121(2), 275 - 82
Characterization of T cell clones derived from lymph nodes and lungs of Pseudomonas aeruginosa-susceptible and resistant mice following immunization with heat-killed bacteria; Kondratieva TK et al.; Pseudomonas aeruginosa-resistant BALB/c and susceptible C57Bl/6 (B6) mice were immunized with heat-killed Pseudomonas either in the foot pad or via the trachea, and panels of Pseudomonas-specific T cell clones were developed from lymph nodes and lungs . All clones from either strain, whether of lymph node or lung origin, were CD3+CD4+CD8-TCRalphabeta+ . The efficacy of cloning from lymph node cells was comparable between BALB/c and B6 mice . All lymph node BALB/c clones proliferated in response to Pseudomonas antigen in a dose-dependent manner, and this response was MHC class II-restricted . Vigorous proliferation by a considerable proportion of B6 T cell clones occurred in the absence of specific antigen . Lymph node clones from either strain could be categorized as either Th1 or Th0 on the basis of interferon-gamma (IFN-gamma)/IL-4 production . In either mouse strain the efficacy of cloning from lung tissue was substantially lower than from lymph nodes, but the efficacy of cloning from BALB/c compared with B6 lungs was higher . Four lung T cell clones from BALB/c and two from B6 mice were expanded for further analyses, and an interstrain difference was observed in cytokine production . Both B6 lung T cell clones were Th1-like and produced IFN-gamma but not IL-4 and IL-10, whereas four BALB/c lung T cell clones were Th2-like and produced IL-4 and IL-10 but not IFN-gamma . These observations suggest that differences in the CD4+ Th response in the lung may contribute to differences among inbred mouse strains in the level of resistance to bronchopulmonary Pseudomonas infection.

Vaccine, 2000 Sep 15, 19(2-3), 348 - 57
Catalase immunization from Pseudomonas aeruginosa enhances bacterial clearance in the rat lung; Thomas LD et al.; Pseudomonas aeruginosa is a common cause of infection in immunocompromised patients and is the major contributor to morbidity in individuals with cystic fibrosis (CF) . The antibiotic resistance shown by this pathogen and morbidity in patients with chronic infection has encouraged investigations into the development of a vaccine . This study reports the purification of a 60 kDa protein, isolated from a mucoid strain of P . aeruginosa, identified by amino acid sequence analysis as the catalase protein (KatA) . A rat model of acute P . aeruginosa respiratory infection was used to investigate the immunogenicity of KatA and determine the potential of mucosal immunization with KatA to protect against infection . Immunization regimens compared a single intra-Peyer's patch (IPP) immunization with an IPP primary inoculation followed by an intratracheal boost to the lungs . Mucosal immunization with KatA resulted in significant pulmonary clearance of both homologous (p<0.001) and heterologous (p<0.05) strains of P . aeruginosa . Both immunization regimens enhanced bacterial clearance, increased the rate of recruitment of phagocytes to the bronchoalveoli and induced KatA-specific antibody . However, the regimen that included a boost induced a more effective immune response that also resulted in better clearance of P . aeruginosa from the lungs . Mucosal immunization induced KatA- specific antibodies in the serum and the bronchoalveolar lavage, and KatA-specific lymphocyte proliferation in vitro in cells isolated from the mesenteric lymph nodes of immunized rats . The data presented suggests that KatA has the potential to afford a protective immune response against pulmonary infection by P . aeruginosa

Int J Antimicrob Agents, 2000 Aug, 15(4), 265 - 9
Distribution and antibiotic resistance of isolates from lower respiratory tract and blood cultures from patients in three Italian intensive care units: a 2-year comparison; Nicoletti G et al.; The distribution and antibiotic resistance of major pathogens isolated from patients in ICUs were studied by three Italian microbiological laboratories . Consecutive aerobic strains were collected over two different time periods from protected brushing bronchoscopy, broncho-alveolar lavage and blood cultures . A total of 420 strains were isolated during the first period (47.3% gram-negative and 52.7% gram-positive) and 412 over the second period (50.5% gram-negative and 49.5% gram-positive) . Pseudomonas aeruginosa was the most frequently isolated organism from the respiratory tract followed by Staphylococcus aureus . Methicillin resistance was 47.9 and 44.5% in S . aureus and 63.0 and 65.1% in coagulase-negative staphylococci over the two periods . No glycopeptide-resistance was found in gram-positive organisms . Ceftazidime-resistance in Klebsiella pneumoniae was very high.

Int J Antimicrob Agents, 2000 Aug, 15(4), 257 - 63
High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3'-phosphotransferase with high affinity for amikacin; Torres C et al.; This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside . Amikacin-resistant P . aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI . The enzyme from P . aeruginosa PA40 was purified to > 70% homogeneity . The Km of amikacin for this enzyme was 1.4 microM, the Vmax/Km ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps . Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed.

Z Naturforsch {C}, 2000 May-Jun, 55(5-6), 347 - 54
Metal binding to Pseudomonas aeruginosa azurin: a kinetic investigation; Naro F et al.; The interaction between azurin from Pseudomonas aeruginosa and Ag(I), Cu(II), Hg(II), was investigated as a function of protein state, i.e . apo-, reduced and oxidised azurin . Two different metal binding sites, characterized by two different spectroscopic absorbancies, were detected: one is accessible to Ag(I) and Cu(II) but not to Hg(II); the other one binds Ag(I) and Hg(II) but not copper . When added in stoichiometric amount, Ag(I) shows high affinity for the redox center of apo-azurin, to which it probably binds by the -SH group of Cys112; it can displace Cu(I) from reducedazurin, while it does not bind to the redox center of oxidizedazurin . Kinetic experiments show that Ag(I) binding to the reduced form is four times faster than binding to the apo-form . This result suggests that metal binding requires a conformational rearrangement of the active site of the azurin . Interaction of Ag(I) or Hg(II) ions to the second metal binding site, induces typical changes of UV spectrum and quenching of fluorescence emission.

Acta Paediatr Taiwan, 2000 Mar-Apr, 41(2), 98 - 100
Pyogenic liver abscess caused by Pseudomonas aeruginosa in a previously healthy child: report of one case; Lo WT et al.; Pyogenic liver abscess (PLA), a very uncommon liver disease in the normal pediatric group is often associated with immunocompromised conditions . Pseudomonas aeruginosa has long been regarded as a relatively rare pathogen of PLA, especially in patients without underlying problems . A previously healthy one-year-and-seven-month-old boy who had symptoms of fever, vomiting and diarrhea got a liver abscess at right hepatic lobe which was confirmed by abdominal ultrasound and computed tomography (CT) diagnoses . Ultrasound-guided percutaneous aspiration of liver abscess was done soon after the confirmation . The culture result of aspirate grew P . aeruginosa . The patient received a 4-week course of adequate antibiotics treatment after the aforementioned aspiration procedure . In addition, a series of ultrasounds were performed to follow the resolution of abscess during the treatment period . The immune function tests of the patient were within normal ranges . Finally, the lesion resolved completely without leaving any complication.

ASAIO J, 2000 Jul-Aug, 46(4), 444 - 7
Pyrogen retention by the Asahi APS-650 polysulfone dialyzer during in vitro dialysis with whole human donor blood; Linnenweber S et al.; The purpose of this study was to test the pyrogen permeability of the new Asahi polysulfone APS 650 (APS) dialyzer membrane with a high permeability for middle molecules (up to 40 kDa) in comparison with the high-flux Fresenius polysulfone F60S (F60S) membrane . Dialyzers were tested in parallel in vitro dialysis experiments with whole human donor blood in the blood compartment and contaminated bicarbonate dialysate in the dialysate compartment . Dialysate was contaminated by a filtrate (0.45 microm) of a Pseudomonas aeruginosa culture in bicarbonate dialysate . The production of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in whole blood samples taken from the in vitro dialysis system was used to detect the passage of cytokine inducing bacterial substances derived from P . aeruginosa across the two highflux polysulfone membranes . Compared with a sterile control period at the beginning of each experiment (n = 5), the TNFalpha inducing activity in the dialysate increased from (mean +/- SEM) 42 +/- 12 pg/ml to 1,288 +/- 356 pg/ml with F60S dialyzers and from 37 +/- 10 pg/ml to 928 +/- 249 pg/ml with APS dialyzers 30 minutes after the dialysate was contaminated . The IL-1beta inducing activity in the dialysate increased similarly . In the presence of this significant contamination in the dialysate, whole blood circulating in the blood compartments for 60 minutes was not stimulated to produce increased amounts of TNFalpha or IL-1beta with neither of the two tested membranes . We conclude that F60S and APS membranes are equal in their ability to prevent the passage of cytokine inducing bacterial substances from highly contaminated dialysate into the patients' blood during hemodialysis.

Bull Soc Belge Ophtalmol, 2000, 276, 53 - 6
Spontaneous corneal perforation and endophthalmitis in Pseudomonas aeruginosa infection in a ventilated patient: a case report; Wynants S et al.; We report a case of Pseudomonas keratitis and endophthalmitis after inoculation from the respiratory tract in a mechanically ventilated patient . In these (semi)comatose and more vulnerable patients, colonisation of the upper respiratory tract by Pseudomonas occurs frequently, and this can lead to inoculation of the eyes . Emphasis lies on careful prevention of ocular inoculation and aggressive therapy as soon as keratitis is noticed.

Appl Environ Microbiol, 2000 Aug, 66(8), 3535 - 42
Characterization of an isolate that uses vinyl chloride as a growth substrate under aerobic conditions; Verce MF et al.; An aerobic enrichment culture was developed by using vinyl chloride (VC) as the sole organic carbon and electron donor source . VC concentrations as high as 7.3 mM were biodegraded without apparent inhibition . VC use did not occur when nitrate was provided as the electron acceptor . A gram-negative, rod-shaped, motile isolate was obtained from the enrichment culture and identified based on biochemical characteristics and the sequence of its 16S rRNA gene as Pseudomonas aeruginosa, designated strain MF1 . The observed yield of MF1 when it was grown on VC was 0.20 mg of total suspended solids (TSS)/mg of VC . Ethene, acetate, glyoxylate, and glycolate also served as growth substrates, while ethane, chloroacetate, glycolaldehyde, and phenol did not . Stoichiometric release of chloride and minimal accumulation of soluble metabolites following VC consumption indicated that the predominant fate for VC is mineralization and incorporation into cell material . MF1 resumed consumption of VC after at least 24 days when none was provided, unlike various mycobacteria that lost their VC-degrading ability after brief periods in the absence of VC . When deprived of oxygen for 2.5 days, MF1 did not regain the ability to grow on VC, and a portion of the VC was transformed into VC-epoxide . Acetylene inhibited VC consumption by MF1, suggesting the involvement of a monooxygenase in the initial step of VC metabolism . The maximum specific VC utilization rate for MF1 was 0.41 micromol of VC/mg of TSS/day, the maximum specific growth rate was 0.0048/day, and the Monod half-saturation coefficient was 0.26 microM . A higher yield and faster kinetics occurred when MF1 grew on ethene . When grown on ethene, MF1 was able to switch to VC as a substrate without a lag . It therefore appears feasible to grow MF1 on a nontoxic substrate and then apply it to environments that do not exhibit a capacity for aerobic biodegradation of VC.

Appl Environ Microbiol, 2000 Aug, 66(8), 3262 - 8
Rhamnolipid-induced removal of lipopolysaccharide from Pseudomonas aeruginosa: effect on cell surface properties and interaction with hydrophobic substrates; Al-Tahhan RA et al.; Little is known about the interaction of biosurfactants with bacterial cells . Recent work in the area of biodegradation suggests that there are two mechanisms by which biosurfactants enhance the biodegradation of slightly soluble organic compounds . First, biosurfactants can solubilize hydrophobic compounds within micelle structures, effectively increasing the apparent aqueous solubility of the organic compound and its availability for uptake by a cell . Second, biosurfactants can cause the cell surface to become more hydrophobic, thereby increasing the association of the cell with the slightly soluble substrate . Since the second mechanism requires very low levels of added biosurfactant, it is the more intriguing of the two mechanisms from the perspective of enhancing the biodegradation process . This is because, in practical terms, addition of low levels of biosurfactants will be more cost-effective for bioremediation . To successfully optimize the use of biosurfactants in the bioremediation process, their effect on cell surfaces must be understood . We report here that rhamnolipid biosurfactant causes the cell surface of Pseudomonas spp . to become hydrophobic through release of lipopolysaccharide (LPS) . In this study, two Pseudomonas aeruginosa strains were grown on glucose and hexadecane to investigate the chemical and structural changes that occur in the presence of a rhamnolipid biosurfactant . Results showed that rhamnolipids caused an overall loss in cellular fatty acid content . Loss of fatty acids was due to release of LPS from the outer membrane, as demonstrated by 2-keto-3-deoxyoctonic acid and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and further confirmed by scanning electron microscopy . The amount of LPS loss was found to be dependent on rhamnolipid concentration, but significant loss occurred even at concentrations less than the critical micelle concentration . We conclude that rhamnolipid-induced LPS release is the probable mechanism of enhanced cell surface hydrophobicity.

Mayo Clin Proc, 1999 Oct, 74(10), 1030 - 7
The fluoroquinolones; Walker RC; The quinolones are broad-spectrum antibacterial agents that have a novel mechanism of action . As synthetic compounds, these agents have been developed extensively to optimize antimicrobial activity, pharmacokinetic properties, and drug safety . Although earlier quinolones were effective only in the genitourinary and gastrointestinal tracts and only had activity against aerobic gram-negative bacteria, newer quinolones have wider potential applications and a broader spectrum of activity . Some of the newer quinolones will have a role in the treatment of community-acquired pneumonia and intra-abdominal infections . Ciprofloxacin remains the most potent quinolone against Pseudomonas aeruginosa . Among the quinolones, important differences exist in renal and hepatic elimination and dose-adjustment regimens . Although there are many Food and Drug Administration-approved indications for some of the newer quinolones, the quinolones are the drug of choice for only a few infections . Quinolone-resistant bacteria are being increasingly identified and emerge under selective pressure created by extensive use.

Vestn Oftalmol, 2000 May-Jun, 116(3), 35 - 7
{Therapeutic algorithms in infectious ulcers of cornea}; Maichuk IuF; Based on the experience gained at Department of Infections and Allergic Diseases of the Eyes and recent publications, the author proposes protocols of drug therapy of the major infectious ulcerative diseases of the cornea: herpetic ulcer, ulcer caused by Pseudomonas aeruginosa, gonococci, staphylococci, fungi, and acanthamedian keratitis . The authors emphasizes that the treatment of ulcers of the cornea should be complex, including specific therapy (corresponding to the identified agent or the agent most probable as judged from the clinical picture) and pathogenetic therapy (metabolic, antiallergic, antiinflammatory, immunomodulating, hypotensive) . If corneal ulcer is rapidly recognized and forced therapy started, good results can be attained even in such a fulminant infection as those caused by Pseudomonas aeruginosa or gonococci . On the other hand, therapy of keratomycosis and acanthamebian keratitis remains a problem.

Support Care Cancer, 2000 Jul, 8(4), 293 - 301
Ceftriaxone versus beta-lactams with antipseudomonal activity for empirical, combined antibiotic therapy in febrile neutropenia: a meta-analysis; Furno P et al.; The object of this work was to compare the efficacy of antibiotic combinations including ceftriaxone with that of combinations including an antipseudomonal beta-lactam for the empirical treatment of febrile neutropenia in cancer patients . We identified all published randomised trials comparing two antibiotic combinations differing only in the beta-lactam, being ceftriaxone in one treatment group and an antipseudomonal beta-lactam in the other . The quality of individual trials was formally evaluated . A meta-analysis was performed using the Peto-modified Mantel-Haenszel method for combining binary data . Primary analysis was done, for both febrile episodes and bacteraemic episodes, using failure of empirical antibiotic treatment defined as modification of the initial allocated regimen or death during treatment . Secondary analysis was done using death from any cause in the two treatment groups . Data relating to 1,537 febrile neutropenic episodes recorded in eight randomised clinical trial were pooled s . Overall, there were 256 treatment failures out of 782 febrile episodes treated with ceftriaxone-containing combinations (32.7%), and 243 out of 755 treated with antipseudomonal beta-lactam regimens (32.1%) . The pooled odds ratio of failure for ceftriaxone-containing combinations for febrile episodes was 1.04, with the 95% confidence interval ranging from 0.84 to 1.29, and that for bacteraemic episodes was 0.93 (95% confidence interval 0.58-1.49) . With regard to overall mortality, there were 54 deaths among 782 febrile episodes treated with ceftriaxone-containing combinations (6.9%) and 62 deaths among 755 febrile episodes treated with antipseudomonal beta-lactam-containing regimens (8.2%) . The pooled odds ratio of death for ceftriaxone regimens was 0.84 (95% confidence interval 0.57-1.24) . Results of this meta-analysis show that in the empirical treatment of febrile neutropenia, antibiotic combinations containing ceftriaxone are as effective as those in which the beta-lactam has specific activity against Pseudomonas aeruginosa, such as ureidopenicillin or ceftazidime.

Surgery, 2000 Aug, 128(2), 339 - 44
Bactericidal and endotoxin neutralizing activity of a peptide derived from Limulus antilipopolysaccharide factor; Weiss CA 3rd et al.; BACKGROUND: Release of lipopolysaccharide (endotoxin, LPS) is a critical inciting event in the development of sepsis syndrome due to gram-negative bacteria, and mortality associated with this entity remains approximately 40% . Limulus anti-LPS factor (LALF) is a naturally occurring horseshoe crab derived protein that, unlike antibiotics, is both bactericidal for gram-negative bacteria and capable of neutralizing LPS . We hypothesized that a peptide derived from the active domain of LALF (LALF #28-54) would exhibit potent biologic activity similar to that of LALF itself and could potentially be useful as a therapeutic agent . METHODS: The effects of LALF, synthetic peptide LALF #28-54, polymyxin B (PmB), and a biologically inactive synthetic peptide were examined in several models . In vitro bactericidal activity was determined against Pseudomonas aeruginosa, and LPS-neutralizing capacity was determined via inhibition of LPS-induced tumor necrosis factor-alpha (TNF-alpha) secretion by RAW 264.7 cells . In vivo biologic activity was determined via pretreatment following which P aeruginosa endotoxemia or bacteremia was induced; serum TNF-alpha levels, bacterial clearance, and survival were assessed . RESULTS: LALF and LALF #28-54 exhibited potent in vitro bactericidal and LPS-neutralizing activity comparable to PmB (P <.01) . However, although LALF #28-54 diminished systemic TNF-alpha production and aided bacterial clearance similar to that observed for LALF (P <.01), it did not provide significant protective capacity (P >.1) . CONCLUSIONS: These data demonstrate that peptide LALF #28-54 retained the LPS-neutralizing and bactericidal biologic activity of LALF but failed to protect during overwhelming P aeruginosa bacteremia, perhaps due to short serum half-life.

Rev Esp Quimioter, 2000 Jun, 13(2), 187 - 92
{Risk factors and prognosis of nosocomial pneumonia due to gram-negative bacteria in a general hospital}; Martinez B et al.; Nosocomial pneumonia due to Gram-negative bacteria is one of the most important infections because of its high frequency, morbidity and mortality . The objective of this study was to determine the risk factors and prognosis for nosocomial pneumonia caused by Gram-negative bacteria . A group of 50 patients with nosocomial pneumonia due to Gram-negative bacteria were studied in a prospective, consecutive manner and compared with another group of 50 patients with similar characteristics but without infection . The diagnostic criteria, acquisition, previous infections, prognosis of the underlying disease, the initial severity of the clinical situation, presence of complications, type and evolution of antibiotic treatment were adjusted according to the criteria in the literature . Univariate and multivariate statistical analysis of the results was carried out . The risk factors found included the following: male sex, high-risk hospital units, nosocomial acquisition and previous manipulation with intubation and mechanical ventilation, previous pulmonary infections, and the use of wide-spectrum antibiotics in the six weeks prior to the study . The most isolated Gram-negative bacterium was Pseudomonas aeruginosa (32%), followed by polymicrobial flora (18%) . Bacteriemia was found in 30% of the cases . Mortality was 24%, with the factors significantly associated with a poor prognosis being a serious underlying disease, a clinically critical situation, previous surgery, complications, Gram-negative bacteria, use of wide-spectrum antibiotics in the six months before the study, and advanced age . The mortality of the group was 8% . It was concluded that knowledge of the risk factors and prognosis of nosocomial pneumonia due to Gram-negative bacteria is of high importance to improve treatment and decrease morbidity and mortality.

Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 8815 - 21
Plants and animals share functionally common bacterial virulence factors; Rahme LG et al.; By exploiting the ability of Pseudomonas aeruginosa to infect a variety of vertebrate and nonvertebrate hosts, we have developed model systems that use plants and nematodes as adjuncts to mammalian models to help elucidate the molecular basis of P . aeruginosa pathogenesis . Our studies reveal a remarkable degree of conservation in the virulence mechanisms used by P . aeruginosa to infect hosts of divergent evolutionary origins.

Crit Care Med, 2000 Jul, 28(7), 2397 - 405
Effects of inhaled nitric oxide in a rat model of Pseudomonas aeruginosa pneumonia; Webert KE et al.; OBJECTIVE: Antimicrobial effects of nitric oxide (NO) have been demonstrated in vitro against a variety of infectious pathogens, yet in vivo evidence of a potential therapeutic role for exogenous NO as an antimicrobial agent is limited . Thus, we assessed the effects of inhaled NO on pulmonary infection, leukocyte infiltration, and NO synthase (NOS) activity in a rat model of Pseudomonas aeruginosa pneumonia . DESIGN: Controlled animal study . SETTING: Research laboratory of an academic institution . SUBJECTS: Male Sprague-Dawley rats . INTERVENTIONS: After intratracheal instillation of either P . aeruginosa or saline (sham), rats were randomly exposed to either 40 ppm of inhaled NO or room air (RA) for 24 hrs before they were killed . MEASUREMENTS AND MAIN RESULTS: Inhaled NO in pneumonia rats markedly reduced pulmonary bacterial load (0.02+/-0.01% vs . 0.99+/-0.59% of bacterial input in pneumonia with room air, p < .05) and pulmonary myeloperoxidase activity, a marker of leukocyte infiltration (21.7+/-3.8 vs . 55.0+/-8.1 units in pneumonia with room air, p < .05), but had no effect on systemic hemodynamics or gas exchange . Pneumonia was associated with enhanced pulmonary NOS activity (8.8+/-2.4 vs . 0.2+/-0.1 pmol citrulline/min/mg protein in sham, p < .01) and increased plasma levels of nitrites/nitrates (NOx-; 45+/-7 vs . 16+/-3 micromol/L in sham, p < .01) . Inhaled NO therapy attenuated the pneumonia-induced increase in pulmonary calcium-independent NOS activity (p < .05) and markedly increased plasma NOx- levels . Exposure of P . aeruginosa in culture to 40 ppm of ambient NO confirmed a delayed antibacterial effect of NO in vitro . CONCLUSIONS: Inhaled NO has an important antibacterial effect both in vitro and in vivo against P . aeruginosa and is associated with reduced pulmonary leukocyte infiltration in vivo . These results in a rat model of P . aeruginosa pneumonia suggest that future studies should address the possible clinical effects of inhaled NO therapy in pneumonia.

Am J Respir Cell Mol Biol, 2000 Aug, 23(2), 121 - 7
Inflammation and infection in naive human cystic fibrosis airway grafts; Tirouvanziam R et al.; Exacerbated inflammation is now recognized as an important component of cystic fibrosis (CF) airway disease . Whether inflammation is part of the basic defect in CF or a response to persistent infection remains controversial . We addressed this question using human fetal tracheal grafts in severe combined immunodeficient mice . This model yields histologically mature, and most importantly, naive CF and non-CF surrogate airways . Significant inflammatory imbalance was found in naive CF airway grafts, including a highly increased intraluminal interleukin 8 content (CF: 10.1 +/- 2.2 ng/ml; non-CF: 1.2 +/- 0.6 ng/ml; P < 0.05) and consistent accumulation of leukocytes in the subepithelial region (P < 0.001) . CF airway grafts were not histologically affected until challenged with Pseudomonas aeruginosa, which provoked: (1) early (before 3 h) and massive leukocyte transepithelial migration, (2) intense epithelial exfoliation, and (3) rapid progression of bacteria toward the lamina propria . In non-CF grafts, these three sets of events were not observed before 6 h . Using a model of naive human airways, we thus demonstrate that before any infection, CF airways are in a proinflammatory state . After infection, the basal inflammatory imbalance contributes to exert severe damage to the mucosa, paving the way for bacterial colonization and subsequent steps of CF airway disease.

J Bacteriol, 2000 Aug, 182(16), 4557 - 63
A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities; Hassett DJ et al.; A Pseudomonas aeruginosa oxyR mutant was dramatically sensitive to H(2)O(2), despite possessing wild-type catalase activity . Oxygen-dependent oxyR phenotypes also included an inability to survive aerobic serial dilution in Luria broth and to resist aminoglycosides . Plating the oxyR mutant after serial dilution in its own spent culture supernatant, which contained the major catalase KatA, or under anaerobic conditions allowed for survival . KatA was resistant to sodium dodecyl sulfate, proteinase K, pepsin, trypsin, chymotrypsin and the neutrophil protease cathepsin G . When provided in trans and expressed constitutively, the OxyR-regulated genes katB, ahpB, and ahpCF could not restore both the serial dilution defect and H(2)O(2) resistance; only oxyR itself could do so . The aerobic dilution defect could be complemented, in part, by only ahpB and ahpCF, suggesting that the latter gene products could possess a catalase-like activity . Aerobic Luria broth was found to generate approximately 1.2 microM H(2)O(2) min(-1) via autoxidation, a level sufficient to kill serially diluted oxyR and oxyR katA bacteria and explain the molecular mechanism behind the aerobic serial dilution defect . Taken together, our results indicate that inactivation of OxyR renders P . aeruginosa exquisitely sensitive to both H(2)O(2) and aminoglycosides, which are clinically and environmentally important antimicrobials.

J Bacteriol, 2000 Aug, 182(16), 4545 - 56
AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide; Howell ML et al.; In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa . The ankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins . The location of ankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB . Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm . Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions . Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is approximately 65% alpha-helical . RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katB and ankB are part of a small operon whose transcription is induced dramatically by H(2)O(2), and controlled by the global transactivator OxyR . Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of an ankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles . The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H(2)O(2), phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB . Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H(2)O(2) detoxification.

J Bacteriol, 2000 Aug, 182(16), 4533 - 44
Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF; Ochsner UA et al.; Pseudomonas aeruginosa possesses an extensive armament of genes involved in oxidative stress defense, including katB-ankB, ahpB, and ahpC-ahpF . Transcription of these genes was regulated in response to H(2)O(2), paraquat, or organic peroxides . Expression of katB-lacZ and the observed KatB catalase levels in P . aeruginosa PAO1 were induced up to 250-fold after exposure to oxidative stress-generating compounds . Also, ahpB-lacZ and ahpC-lacZ expression was 90- and 3-fold higher, respectively, upon exposure to paraquat . The dose- and time-response curves revealed that 1 microM paraquat was sufficient for half-maximal activation of each reporter fusion within 5 min of exposure . Expression of these genes was not observed in a DeltaoxyR mutant, indicating that OxyR was essential for this response . The transcriptional start sites of katB-ankB, ahpB, and ahpC-ahpF were mapped, putative OxyR-binding sites were identified upstream of the -35 promoter elements, and direct binding of purified OxyR protein to these target promoters was demonstrated . The oxyR mutant was hypersusceptible to oxidative stress-generating agents, including H(2)O(2) and paraquat, in spite of total KatA catalase activity being comparable to that of the wild type . The oxyR phenotype was fully complemented by a plasmid containing the oxyR gene, while any of the katB, ahpB, or ahpCF genes alone resulted in only marginal complementation . Increased katB-lacZ expression and higher KatB catalase levels were detected in a DeltaahpCF background compared to wild-type bacteria, suggesting a compensatory function for KatB in the absence of AhpCF . In P . aeruginosa, oxyR is located upstream of recG, encoding a putative DNA repair enzyme . oxyR-lacZ and recG-lacZ reporter activities and oxyR-recG mRNA analysis showed that oxyR and recG are organized in an operon and expressed constitutively with regard to oxidative stress from a single promoter upstream of oxyR . Mutants affected in recG but not oxyR were dramatically impaired in DNA damage repair as measured by sensitivity to UV irradiation . In conclusion, we present evidence that the oxyR-recG locus is essential for oxidative stress defense and for DNA repair.

J Bacteriol, 2000 Aug, 182(16), 4453 - 7
Identification of the Pseudomonas aeruginosa glmM gene, encoding phosphoglucosamine mutase; Tavares IM et al.; A search for a potential algC homologue within the Pseudomonas aeruginosa PAO1 genome database has revealed an open reading frame (ORF) of unknown function, ORF540 in contig 54 (July 1999 Pseudomonas genome release), that theoretically coded for a 445-amino-acid-residue polypeptide (I . M . Tavares, J . H . Leitao, A . M . Fialho, and I . Sa-Correia, Res . Microbiol . 150:105-116, 1999) . The product of this gene is here identified as the phosphoglucosamine mutase (GlmM) which catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the formation of the cell wall precursor UDP-N-acetylglucosamine . The P . aeruginosa gene has been cloned into expression vectors and shown to restore normal peptidoglycan biosynthesis and cell growth of a glmM Escherichia coli mutant strain . The GlmM enzyme from P . aeruginosa has been overproduced to high levels and purified to homogeneity in a six-histidine-tagged form . Beside its phosphoglucosamine mutase activity, the P . aeruginosa enzyme is shown to exhibit phosphomannomutase and phosphoglucomutase activities, which represent about 20 and 2% of its GlmM activity, respectively.

Am J Otol, 2000 Jul, 21(4), 462 - 7
Granular myringitis: is it a surgical problem?
El-Seifi A, Fouad B.
OBJECTIVE: An attempt to settle the controversies associated with granular myringitis (GM) including incidence, etiology, pathology, presentation, relation to chronic otitis media, and treatment . STUDY DESIGN: Retrospective . SETTING: Tertiary referral center and private otology practice . PATIENTS: 94 patients presenting with GM over 28 years . INTERVENTION: Diagnosis by otoscopy, audiometry, radiology, and bacteriology; long-term follow-up (6 months to 12 years); assessment of treatment results . MAIN OUTCOME MEASURES: The pathologic states of the affected tympanic membranes were studied in both active and quiescent stages . The results of conservative versus surgical management were evaluated . RESULTS: The disease presents with chronic painless otorrhea, normal hearing and mastoid pneumatization, and granular areas, which may be patchy, diffuse, or segmental . The latter is the most frequent and is most commonly posterosuperior . The infecting organism is Pseudomonas aeruginosa . The pathologic process affects all drum layers and can cause a perforation . The most important predisposing factor is disturbed epithelial migration, which may be exaggerated by eustachian tube dysfunction . Of 26 cases treated conservatively, none healed without recurrence . Of 48 cases treated surgically, there were 2 recurrences . CONCLUSIONS: Pathologically, the disease affects all drum layers . It presents with an active stage, which may be misdiagnosed as chronic otitis media or cholesteatoma, and a quiescent stage when it may be overlooked . Although distinct from chronic otitis media, it can cause a perforation . The disease responds readily to medical treatment, but recurrence is common . Radical surgery offers a curative measure in refractory cases.

Klin Khir, 1993, (2), 13 - 5
{The clinico-diagnostic significance of the general antiprotease capacity of the blood in assessing endotoxicosis in suppurative-inflammatory diseases of the abdominal cavity organs}; Protsiuk AV et al.; The views upon the value of general antiprotease capacity of the blood in evaluation of endotoxicosis in purulent-inflammatory processes of the abdominal cavity are presented . One hundred and thirty one patient with diffuse peritonitis, acute pancreatitis and obstructive jaundice was examined . A role of bacterial proteases in exhaustion of the inhibitors at the example of Pseudomonas aeruginosa strain has been established . Use of protease inhibitors (contrykal etc.) only for strict indications is substantiated.

Acta Paediatr Taiwan, 1999 Jul-Aug, 40(4), 233 - 6
Community-acquired Pseudomonas aeruginosa bacteremia and sepsis in previously healthy infants; Wu BY et al.; Pseudomonas aeruginosa bacteremia, or sepsis, often occurs in hospitals, affecting mainly children with underlying problems . However, it can also appear in communities, and affects infants and children without underlying diseases . We report eight cases of Pseudomonas aeruginosa bacteremia, or sepsis, in previously healthy infants over a three-year period . All patients were less than twelve months old and the majority presented with sepsis, diarrhea, ecthyma gangrenosum, and neutropenia . The infection route may have been the gastrointestinal tract . Concomitant gastrointestinal infections may have played a role in pathogenesis.

Ned Tijdschr Geneeskd, 2000 Jun 24, 144(26), 1261 - 6
{Choice of ear drops in chronic otorrhea}; Mylanus EA et al.; In chronic otitis, the use of ear drops has certain advantages over the use of systemic antibiotics . The choice of ear drop depends on the condition of the eardrum, microbial pathogens present and the efficacy of the components of the ear drop . Ototoxicity, contact allergy and the development of bacterial resistance have to be taken into account . Ototoxicity is a rare complication of the application of ear drops, most often described when aminoglycosides were applied . Contact allergy is also most often seen in aminoglycoside-containing eardrops . Evaluation of ear swabs demonstrated a 5% resistance of Pseudomonas aeruginosa to ciprofloxacin . The appearance of resistant strains may impede systemic use of fluoroquinolones . Therefore, this class of antibiotics should be considered as reserve medication only . The first choice in local application of antiseptics in case of an open eardrum is aluminium acetotartrate 1.2% and, of a combination preparation, bacitracin-colistin-hydrocortisone . In case of a closed eardrum (external otitis) aluminium acetotartrate 12%--combination preparations with corticosteroids are advised against in these cases.

Cochrane Database Syst Rev . 2000;(3):CD002203.
Macrolide antibiotics for cystic fibrosis; Southern KW et al.; BACKGROUND: Cystic Fibrosis is characterised by chest infection, the antibiotic treatment of which has significantly improved the outlook for people with this condition . The unusual nature of organisms that infect the chest of individuals with cystic fibrosis has restricted antibiotic choice . In particular the bacteria, Pseudomonas aeruginosa, is resistant to nearly all antibiotics that can be taken by mouth . There is laboratory evidence and evidence from other disease processes that macrolide antibiotics, whilst not directly active against Pseudomonas aeruginosa, may have indirect actions against this bacteria . OBJECTIVES: This review aimed to test the hypotheses that macrolide antibiotics; 1) Improve clinical status compared to placebo or another antibiotic 2) do not have unacceptable adverse effects If benefit was demonstrated, we aimed to assess the optimal type, dose and duration of macrolide therapy . SEARCH STRATEGY: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group specialist trials register which comprises references identified from comprehensive electronic database searches, handsearching relevant journals and handsearching abstract books of conference proceedings . In addition, Principal Investigators, known to work in the field and previous authors were contacted for unpublished or follow up data . Pharmaceutical companies, that manufacture macrolide antibiotics, were approached . SELECTION CRITERIA: Randomised controlled trials, published or unpublished, of macrolide compared to placebo, another class of antibiotic or another macrolide . Studies which compare regimes of the same macrolide at different doses will also be included . DATA COLLECTION AND ANALYSIS: No completed randomised controlled trials were identified . MAIN RESULTS: Three open studies excluded . Four ongoing randomised controlled trials were identified . No completed randomised controlled trials were identified . REVIEWER'S CONCLUSIONS: At present, there are no randomised controlled trials to evaluate the use of macrolide antibiotics for the treatment of chest infection in people with cystic fibrosis . Such trials, with clear outcome measures, are needed to properly evaluate this potentially useful treatment for cystic fibrosis.

J Biomed Mater Res, 2000 Oct, 52(1), 88 - 94
Well-defined sulfobetaine-based statistical copolymers as potential antibioadherent coatings; Lowe AB et al.; The potential use of novel poly(sulfobetaine) copolymers as antibioadherent coatings was investigated using Pseudomonas aeruginosa as a model microorganism and human macrophages and 3T3 mouse embryonic fibroblasts . Two well-defined statistical copolymers with narrow molecular weight distributions were prepared by group transfer copolymerization of n-butyl methacrylate (nBuMA) with either 10 or 30 mol % 2-(dimethylamino)ethyl methacrylate (DMAEMA) . Sulfobetainized nBuMA-DMAEMA copolymers (poly{sulfobetaine-stat-nBuMA}) were obtained by treating these precursor polymers with 1,3-propanesultone under mild conditions . Both proton NMR spectroscopy and elemental microanalyses indicated that essentially all the DMAEMA residues were derivatized in both copolymers . Poly(methyl methacrylate) (PMMA) discs were coated with the sulfobetainized nBuMA-DMAEMA copolymers and the bioadherent properties of these coated materials were compared with those of PMMA . Statistically significantly fewer (p<.05) bacteria, macrophages, and fibroblasts adhered to the poly(sulfobetaine-stat-nBuMA)-coated PMMA than to the uncoated PMMA . The poly(sulfobetaine-stat-nBuMA) copolymer containing the higher proportion (30 mol %) sulfobetainized DMAEMA residues proved to be the more effective antibioadherent coating . The antibioadherent properties of these coating materials may allow the cost-effective production of dirt-resistant, easy to clean work surfaces, bioinert coatings for medical devices, and antifouling coatings for marine, agricultural, and industrial applications .

J Immunol, 2000 Aug 1, 165(3), 1513 - 9
Urokinase receptor-deficient mice have impaired neutrophil recruitment in response to pulmonary Pseudomonas aeruginosa infection; Gyetko MR et al.; Leukocytes express both urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87) . Evidence in vitro has implicated uPAR as a modulator of beta2 integrin function, particularly CR3 (CD11b/CD18, Mac-1) . Pseudomonas aeruginosa infection has been demonstrated to recruit neutrophils to the pulmonary parenchyma by a beta2 integrin-dependent mechanism . We demonstrate that mice deficient in uPAR (uPAR-/-) have profoundly diminished neutrophil recruitment in response to P . aeruginosa pneumonia compared with wild-type (WT) mice . The requirement for uPAR in neutrophil recruitment is independent of the serine protease uPA, as neutrophil recruitment in uPA-/- mice is indistinguishable from recruitment in WT mice . uPAR-/- mice have impaired clearance of P . aeruginosa compared with WT mice, as demonstrated by CFU and comparative histology . WT mice have diminished neutrophil recruitment to the lung when an anti-CD11b mAb is given before inoculation with the pathogen, while recruitment of uPAR-/- neutrophils is unaffected . We conclude that uPAR is required for the recruitment of neutrophils to the lung in response to P . aeruginosa pneumonia and that this requirement is independent of uPA . Further, we show that uPAR and CR3 act by a common mechanism during neutrophil recruitment to the lung in response to P . aeruginosa . This is the first report of a requirement for uPAR during cellular recruitment in vivo against a clinically relevant pathogen.

Am J Respir Crit Care Med, 2000 Jul, 162(1), 328 - 30
Treatment of nosocomial pneumonia and tracheobronchitis caused by multidrug-resistant Pseudomonas aeruginosa with aerosolized colistin; Hamer DH; Gram-negative bacilli including multidrug-resistant (MDR) Pseudomonas aeruginosa are responsible for a significant proportion of episodes of nosocomial pneumonia . Since the development of new antibiotics with activity against gram-negative organisms has not kept pace with the increase in prevalence of MDR pathogens, there has been renewed interest in antimicrobial agents that had previously been used but had been abandoned because of toxic side effects . This report describes three patients with nosocomial pneumonia or tracheobronchitis due to multiresistant strains of P . aeruginosa for whom aerosolized colistin proved beneficial as supplemental therapy . Aerosolized colistin merits further consideration as a therapeutic intervention for patients with pulmonary infections due to MDR P . aeruginosa.

Biochem J, 2000 Aug 1, 349 Pt 3, 697 - 701
14-3-3 proteins are required for the inhibition of Ras by exoenzyme S; Henriksson ML et al.; 14-3-3 proteins play a regulatory role and participate in both signal transduction and checkpoint control pathways . 14-3-3 proteins bind phosphoserine ligands, such as Raf-1 kinase and Bad, by recognizing the phosphorylated consensus motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where 'Xaa' represents 'any residue', and 'pSer' is 'phosphoserine') . However, 14-3-3 proteins must bind unphosphorylated ligands, such as glycoprotein Ibalpha and Pseudomonas aeruginosa exoenzyme S (ExoS), since it has been suggested that specific residues of 14-3-3 proteins are required for activation of ExoS . Furthermore, an unphosphorylated peptide derived from a phage display library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the same conserved amphipathic groove on the surface of 14-3-3 as the Raf-derived phosphopeptide (pS-Raf-259) . In the present study we identify the interaction site on ExoS for 14-3-3, and show that ExoS and 14-3-3 do indeed interact in vivo . In addition, we show that this interaction is critical for the ADP-ribosylation of Ras by ExoS, both in vitro and in vivo . Loss of the 14-3-3 binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras, and displays reduced killing activity.

Anesteziol Reanimatol, 2000 May-Jun, (3), 41 - 3
{The lipid peroxidation processes and the antioxidant system in the dynamics of an experimental Pseudomonas aeruginosa intoxication}; Morrison VV et al.; Lipid peroxidation and antioxidant defense processes have been studied over the course of experimental Pseudomonas aeruginosa intoxication in albino rats injected with a lethal dose of exotoxin A . The development of intoxication is associated with intensification of lipid peroxidation, manifested by accumulation of malonic dialdehyde and diene conjugates and decreased peroxide resistance of erythrocytes . Superoxide dismutase and catalase activities were inhibited and the concentration of vitamin B dropped . Injection of antioxidant alpha-tocopherol or antihypoxant gutimine at the late stage of intoxication did not notably modify the studied parameters.

Infect Immun, 2000 Aug, 68(8), 4811 - 4
Pseudomonas aeruginosa exoenzyme S induces transcriptional expression of proinflammatory cytokines and chemokines; Epelman S et al.; Pseudomonas aeruginosa infection of cystic fibrosis patients causes lung damage that is substantially orchestrated by cytokines . In this study, multi-gene probe analysis was used to characterize the ability of the P . aeruginosa mitogen, exoenzyme S, to induce proinflammatory and immunoregulatory cytokines and chemokines . Exoenzyme S strongly induced transcription of proinflammatory cytokines and chemokines (tumor necrosis factor alpha, interleukin-1alpha {IL-1alpha}, IL-1beta, IL-6, IL-8, MIP-1alpha, MIP-1beta, MCP-1, RANTES, and I-309), modest transcription of immunoregulatory cytokines (IL-10 and IL-12p40), and weak transcription of Th1 cytokines (IL-2 and gamma interferon) . The response occurred early and subsided without evolving over time . These data suggest that cells responding to exoenzyme S would rapidly express proinflammatory cytokines and chemokines that may contribute to pulmonary inflammation in cystic fibrosis.

Infect Immun, 2000 Aug, 68(8), 4585 - 92
Role of pulmonary alveolar macrophages in defense of the lung against Pseudomonas aeruginosa; Cheung DO et al.; Alveolar macrophages (AM) provide one of the first lines of defense against microbial invasion in the lower airways . The role of AM in the clearance of Pseudomonas aeruginosa in mice after intrapulmonary challenge was evaluated . AM were depleted by intranasal administration of liposome-encapsulated dichloromethylene diphosphonate . At 24 h following the instillation of liposomes, a sublethal dose of P . aeruginosa was inoculated intranasally . Spleen, liver, and lung tissue was then evaluated for viable bacteria and for histopathology . AM depletion of 78 to 88% did not affect the survival rate of infected mice or clearance of P . aeruginosa from the spleen, liver, or lung, compared to the control group, but the mice's susceptibility to Klebsiella pneumoniae was greatly enhanced . The recruitment of neutrophils to the lung was also not affected . Freshly explanted AM were not competent to phagocytose unopsonized P . aeruginosa but were able to phagocytose zymosan particles . Further studies were conducted to assess the in situ phagocytic activities of AM . Three hours after the intranasal instillation of P . aeruginosa or other particles, bronchoalveolar lavage was performed . AM phagocytosis of zymosan particles and latex beads exceeded that of P . aeruginosa . Neutrophils were recruited to the lung in response to a high-dose bacterial challenge . These results suggest that AM do not play an important role in defense of the lung against P . aeruginosa.

Infect Immun, 2000 Aug, 68(8), 4498 - 504
Requirement of the Pseudomonas aeruginosa tonB gene for high-affinity iron acquisition and infection; Takase H et al.; To investigate the contribution of the TonB protein to high-affinity iron acquisition in Pseudomonas aeruginosa, we constructed tonB-inactivated mutants from strain PAO1 and its derivative deficient in producing the siderophores pyoverdin and pyochelin . The tonB mutants could not grow in a free-iron-restricted medium prepared by apotransferrin addition, even though the medium was supplemented with each purified siderophore or with a heme source (hemoglobin or hemin) . The tonB inactivation was shown to make P . aeruginosa unable to acquire iron from the transferrin with either siderophore . Introduction of a plasmid carrying the intact tonB gene restored growth of the tonB mutant of PAO1 in the free-iron-restricted medium without any supplements and restored growth of the tonB mutant of the siderophore-deficient derivative in the medium supplemented with pyoverdin, pyochelin, hemoglobin, or hemin . In addition, animal experiments showed that, in contrast to PAO1, the tonB mutant of PAO1 could not grow in vivo, such as in the muscles and lungs of immunosuppressed mice, and could not kill any of the animals . The in vivo growth ability and lethal virulence were also restored by introduction of the tonB-carrying plasmid in the tonB mutant . These results indicate clearly that the intact tonB gene-and, therefore, the TonB protein encoded by it-is essential for iron acquisition mediated by pyoverdin and pyochelin and via heme uptake in P . aeruginosa and suggest that the TonB-dependent iron acquisition may be essential for P . aeruginosa to infect the animal host.

Antimicrob Agents Chemother, 2000 Aug, 44(8), 2187 - 9
Impact of n-6 polyunsaturated fatty acids on growth of multidrug-resistant Pseudomonas aeruginosa: interactions with amikacin and ceftazidime; Giamarellos-Bourboulis EJ et al.; Twenty-six multidrug-resistant Pseudomonas aeruginosa isolates were exposed over time to 300 microg of gamma-linolenic acid or arachidonic acid per ml or to the combination of both acids at 150 microg/ml each with ceftazidime and amikacin with or without albumin to observe the in vitro interactions of the antibiotics . Antibiotics and albumin were applied at their levels found in serum . Synergy between acids and antibiotics was found against 13 isolates, and it was expressed after 5 h of growth in the presence of albumin . The results indicate that further application in experimental infection models is merited.

Eur J Clin Microbiol Infect Dis, 2000 May, 19(5), 370 - 4
Antimicrobial resistance in European isolates of Pseudomonas aeruginosa . European SENTRY Participants; Fluit AC et al.; Pseudomonas aeruginosa is responsible for a substantial fraction of hospital infections . Twenty-five European university hospitals submitted a total of 1411 Pseudomonas aeruginosa isolates for susceptibility testing during 1997 and 1998 . The isolates showed highest susceptibility to amikacin (87.5%), meropenem (87.3%) and piperacillin/tazobactam (86.8%) . Susceptibility to ciprofloxacin was 73.2% . There was no clear geographical distribution of resistance, although isolates from northwestern Europe tended to be more susceptible than those from southeastern Europe . Isolates that were resistant to one class of antibiotics were also often resistant to at least one other class of antibiotics . Imipenem-resistant isolates were generally not clonally related.

Arch Microbiol, 2000 May-Jun, 173(5-6), 346 - 51
Molecular analysis of an outer membrane protein, MopB, of Methylococcus capsulatus (Bath) and structural comparisons with proteins of the OmpA family; Fjellbirkeland A et al.; The gene encoding a major outer membrane protein (MopB) of the methanotroph Methylococcus capsulatus (Bath) was cloned and sequenced . The cloned DNA contained an open reading frame of 1044 bp coding for a 348-amino-acid polypeptide with a 21-amino-acid leader peptide . Comparative sequence analysis of the predicted amino acid sequence revealed that the C-terminal part of MopB possessed sequences that are conserved in the OmpA family of proteins . The N-terminal half of the protein had no significant sequence similarity to other proteins in the databases, but the predicted secondary structure showed stretches of amphipathic beta-strands typical of transmembrane segments of outer membrane proteins . A region with four cysteines similar to the cysteine-encompassing region of the OprF of Pseudomonas aeruginosa was found toward the C-terminal part of MopB . Results from whole-cell labeling with the fluorescent thiol-reacting reagent 5-iodoacetamidofluorescein indicated a surface-exposed location for these cysteines . A probe consisting of the 3'-end of the mopB gene hybridized to the type I methanotroph Methylomonas methanica S in Southern blots containing DNA from nine methanotrophic strains representing six different genera.

J Bacteriol, 2000 Aug, 182(15), 4366 - 71
Autoregulation of the Pseudomonas aeruginosa protein PtxS occurs through a specific operator site within the ptxS upstream region; Swanson BL et al.; We have previously shown that the Pseudomonas aeruginosa toxA regulatory protein PtxS autoregulates its own synthesis by binding to a 52-bp fragment . The 3' end of the 52-bp fragment is located 58 bp 5' of the ptxS translation start site . We have identified a 14-bp palindromic sequence (TGAAACCGGTTTCA) within the 52-bp fragment . In this study, we used site-directed mutagenesis and promoter fusion experiments to determine if PtxS binds specifically to this palindromic sequence and regulates ptxS expression . We have also tried to determine the roles of specific nucleotides within the palindromic sequence in PtxS binding and ptxS expression . Initial promoter fusion experiments confirmed that the 52-bp fragment does not overlap with the region that carries the ptxS promoter activity . PtxS binding was eliminated upon the deletion of the 14-bp palindromic sequence from the 52-bp fragment . In addition, the deletion of the 14-bp sequence caused a significant enhancement in ptxS expression in the P . aeruginosa strain PAO1 and the ptxS isogenic mutant PAO::ptxS . Mutation of specific nucleotides within the 14-bp sequence eliminated, reduced, or had no effect on PtxS binding . However, mutations of several of these nucleotides produced a significant increase in ptxS expression in both PAO1 and PAO::ptxS . These results suggest that (i) the 14-bp palindromic sequence and specific nucleotides within it play a role in PtxS binding and (ii) deletion of the palindromic sequence or changing of certain nucleotides within it interferes with another mechanism that may regulate ptxS expression.

J Bacteriol, 2000 Aug, 182(15), 4356 - 60
Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa; Whiteley M et al.; The LasR-LasI and RhlR-RhlI quorum-sensing systems are global regulators of gene expression in the opportunistic pathogen Pseudomonas aeruginosa . Previous studies suggest that the RhlR-RhlI system activates expression of rpoS . We constructed merodiploid strains of P . aeruginosa containing the native rpoS gene and an rpoS-lacZ fusion . Studies of lacZ transcription in these strains indicated that rpoS was not regulated by RhlR-RhlI . We also generated an rpoS null mutant . This rpoS mutant showed elevated levels of rhlI (but not rhlR) transcription, elevated levels of the RhlI-generated acylhomoserine lactone quorum-sensing signal, and elevated levels of RhlR-RhlI-regulated gene transcription . These findings indicate that there is a relationship between RpoS and quorum sensing, but rather than the RhlR-RhlI system influencing the expression of rpoS, it appears that RpoS regulates rhlI.

Parasitol Res, 2000 Jun, 86(6), 514 - 20
Vannella sp . harboring Microsporidia-like organisms isolated from the contact lens and inflamed eye of a female keratitis patient; Michel R et al.; Viable Hartmannella sp . and two strains of Vannella sp.--but no Acanthamoebae--multiplied on NN-agar inoculated with pieces of the contact lens from a female keratitis patient . Within the cytoplasm of one Vannella isolate, intracellular parasites could be observed whose earliest stages were developing within the nucleus, resembling those Microsporidia-like parasites seen within Vannella isolated recently from a warm tapwater system . This assumption was also confirmed by electron microscopy . In swabs taken directly from the cornea, Pseudomonas aeruginosa were identified, but they did not yield any growth of amebas in culture . However, cocultivation of parasite-free Vannella strains with the above-mentioned swab matter resulted in infected amebas harboring the same intracellular parasites seen before . This infection could be established only if the corresponding spores were present as infective agents in the swab matter . The successful treatment of the patient with antibiotics supports the assumption that P . aeruginosa was the main cause of the corneal ulceration . The extent to which the Microsporidia-like organisms may have been involved in the development of keratitis remains a matter of discussion.

Acta Otolaryngol, 2000 Mar, 120(3), 363 - 8
Dexamethasone modifies the effect of Pseudomonas aeruginosa exotoxin A on hearing; Takeuchi N et al.; In the present study, the protective effect of dexamethasone was analysed following exposure of the cochlea to Pseudomonas aeruginosa Exotoxin A (PaExoA) . Four groups of albino Sprague-Dawley rats were used . 20 microl saline was instilled through the tympanic membrane into the round window niche (group A, n = 4); 1 microg/20 microl dexamethasone sodium 21-phosphate (dexamethasone) solution was instilled (group B, n = 4); 1 microg/20 microl PaExoA solution was initially instilled followed 1 h later by 20 microl saline (group C, n = 6); and 1 microg/20 microl PaExoA solution was initially instilled followed 1 h later by 1 microg/20 microl dexamethasone solution (group D, n = 6) . Frequency-specific (4, 8, 10, 12, 16 and 20 kHz) auditory brainstem responses (ABR) were used to ascertain the threshold prior to exposure and 1, 2, 3, and 5 days and 1 and 2 weeks afterwards . No threshold change was observed in groups A and B, whereas the animals in groups C and D showed some threshold elevation, that in D being smaller than that in C . There was a significant difference at the frequencies 12, 16 and 20 kHz, 2 and 5 days after exposure . The intensity-latency (I-L) curve showed that in group D the cochlear component almost disappeared at high frequency one week after exposure . Our results indicate that dexamethasone can modify the effect of PaExoA caused by non-specific inflammation.

Acta Otolaryngol, 2000 Mar, 120(3), 350 - 8
Otoprotectant minimizes hearing defects caused by Pseudomonas aeruginosa exotoxin A; Popa R et al.; Exotoxin A, produced by Pseudomonas aeruginosa (PaExoA), penetrates from the middle ear in to the cochlea and causes sensorineural hearing loss (SNHL) . In this investigation we studied electrophysiological changes in the albino rat following instillation of PaExoA and N(G)-nitro-L-arginine methyl ester (L-NAME), a known inhibitor of nitric oxide synthesis, into the middle ear . Hearing thresholds were measured by auditory brainstem response (ABR) technique . Latency/intensity curves were constructed to distinguish between cochlear and conductive components of hearing loss . PaExoA caused damage to cochleae and SNHL, mainly at high frequencies . This impairment was blocked by (L-NAME) . It would appear that nitric oxide may be a significant link in the mechanism of SNHL caused by bacterial toxin . L-NAME acts as an otoprotectant against the deleterious action of PaExoA.

Respirology, 2000 Jun, 5(2), 141 - 5
Antigen uptake and subsequent cell kinetics in bronchus-associated lymphoid tissue; Toyoshima M et al.; OBJECTIVE: Bronchus-associated lymphoid tissue (BALT) plays an important role in the immunological defence of airways . However, the mechanisms of BALT development, antigen sampling, and subsequent cell kinetics remain unclear . To clarify these chronological processes, we used a Pseudomonas aeruginosa-exposed mouse model . METHODOLOGY: In BALB/c and C57BL/6 mice, BALT development was induced by inhalation of heat-killed P . aeruginosa after sensitization with subcutaneous injection of P . aeruginosa in the presence of Freund's complete adjuvant . Subsequently, we chronologically killed these mice who had inhaled PKH26-labelled P . aeruginosa and examined bacterial transport using fluorescence microscopy . The distribution of interleukin-4-positive cells and interferon-gamma-positive cells was studied immunohistochemically . RESULTS: The degree of BALT hyperplasia was greater in sensitized mice than in non-sensitized mice and in BALB/c mice than in C57BL/6 mice . PKH26-labelled bacteria were found in BALT earlier in sensitized mice than in non-sensitized mice . Immunohistochemical studies revealed that interleukin-4-positive cells predominated over interferon-gamma-positive cells in the peripheral areas of lymphoid follicles . CONCLUSION: These results indicate that administered antigens are actively transported into BALT and that sensitized Th2 lymphocytes play an important role in forming and maintaining BALT.

J Biol Chem, 2000 Oct 6, 275(40), 31219 - 25
Crystal structure of pseudomonas aeruginosa lipase in the open conformation . The prototype for family I.1 of bacterial lipases; Nardini M et al.; The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 A resolution . It is the first structure of a member of homology family I.1 of bacterial lipases . The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues . Compared with the "canonical" alpha/beta hydrolase fold, the first two beta-strands and one alpha-helix (alphaE) are not present . The absence of helix alphaE allows the formation of a stabilizing intramolecular disulfide bridge . The loop containing His(251) is stabilized by an octahedrally coordinated calcium ion . On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region . A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains . The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction . Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme.

Chest, 2000 Jul, 118(1), 146 - 55
The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting; Ibrahim EH et al.; STUDY OBJECTIVE: To evaluate the relationship between the adequacy of antimicrobial treatment for bloodstream infections and clinical outcomes among patients requiring ICU admission . DESIGN: Prospective cohort study . SETTING: A medical ICU (19 beds) and a surgical ICU (18 beds) from a university-affiliated urban teaching hospital . PATIENTS: Between July 1997 and July 1999, 492 patients were prospectively evaluated . INTERVENTION: Prospective patient surveillance and data collection . RESULTS: One hundred forty-seven patients (29.9%) received inadequate antimicrobial treatment for their bloodstream infections . The hospital mortality rate of patients with a bloodstream infection receiving inadequate antimicrobial treatment (61.9%) was statistically greater than the hospital mortality rate of patients with a bloodstream infection who received adequate antimicrobial treatment (28.4%; relative risk, 2 . 18; 95% confidence interval {CI}, 1.77 to 2.69; p < 0.001) . Multiple logistic regression analysis identified the administration of inadequate antimicrobial treatment as an independent determinant of hospital mortality (adjusted odds ratio {AOR}, 6.86; 95% CI, 5.09 to 9.24; p < 0.001) . The most commonly identified bloodstream pathogens and their associated rates of inadequate antimicrobial treatment included vancomycin-resistant enterococci (n = 17; 100%), Candida species (n = 41; 95.1%), oxacillin-resistant Staphylococcus aureus (n = 46; 32.6%), coagulase-negative staphylococci (n = 96; 21.9%), and Pseudomonas aeruginosa (n = 22; 10.0%) . A statistically significant relationship was found between the rates of inadequate antimicrobial treatment for individual microorganisms and their associated rates of hospital mortality (Spearman correlation coefficient = 0.8287; p = 0.006) . Multiple logistic regression analysis also demonstrated that a bloodstream infection attributed to Candida species (AOR, 51.86; 95% CI, 24.57 to 109.49; p < 0.001), prior administration of antibiotics during the same hospitalization (AOR, 2.08; 95% CI, 1.58 to 2.74; p = 0.008), decreasing serum albumin concentrations (1-g/dL decrements) (AOR, 1.37; 95% CI, 1.21 to 1.56; p = 0.014), and increasing central catheter duration (1-day increments) (AOR, 1.03; 95% CI, 1.02 to 1.04; p = 0.008) were independently associated with the administration of inadequate antimicrobial treatment . CONCLUSIONS: The administration of inadequate antimicrobial treatment to critically ill patients with bloodstream infections is associated with a greater hospital mortality compared with adequate antimicrobial treatment of bloodstream infections . These data suggest that clinical efforts should be aimed at reducing the administration of inadequate antimicrobial treatment to hospitalized patients with bloodstream infections, especially individuals infected with antibiotic-resistant bacteria and Candida species.

J Biol Chem, 2000 Sep 29, 275(39), 30660 - 7
Steric hindrance regulation of the Pseudomonas aeruginosa amidase operon; Norman RA et al.; Expression of the amidase operon of Pseudomonas aeruginosa is controlled by AmiC, the ligand sensor and negative regulator, and AmiR the transcription antitermination factor activator . We have titrated out AmiC repression activity in vivo by increased AmiR production in trans and shown AmiC regulation of the antitermination activity of AmiR by a steric hindrance mechanism . In the presence of the co-repressor butyramide we have isolated a stable AmiC.AmiR complex . Addition of the inducing ligand acetamide to the complex trips the molecular switch, causing complex dissociation and release of AmiR . The AmiC.AmiR butyramide complex exhibits acetamide-dependent, sequence-specific RNA binding activity and a K(d) of 1.0 nm has been calculated for the AmiR.RNA interaction . The results show that amidase operon expression is controlled by a novel type of signal transduction system in which activity of a site-specific RNA binding activator is regulated via a sequestration mechanism.

FEMS Immunol Med Microbiol, 2000 Aug, 28(4), 313 - 8
Rattus norvegicus: not a model for Aeromonas-associated gastroenteritis in man; Kelleher A et al.; The lack of a suitable animal model of Aeromonas-associated diarrhoea has hampered investigations into Aeromonas pathogenic mechanisms . Hence, a published report that clindamycin-pretreated rats developed signs and symptoms of enteritis following intragastric inoculation of an Aeromonas strain required further investigation . Although we could demonstrate long-term colonisation (>12 days) and histological damage in this animal model with Pseudomonas aeruginosa isolated from patients with chronic diarrhoea, this was not seen with Aeromonas spp . Six Aeromonas strains, selected for their potential virulence and colonising abilities and including the strain from the original report, were either not recovered from stools or were recovered for no longer than 2 days post inoculation . Intestinal histology remained normal . Destruction of bacteria in vivo appeared to be due to immune mechanisms as inoculum strains were not 'suicidal' or unduly sensitive to low pH or clindamycin . This study was, therefore, unable to validate the clindamycin-treated rat model as a useful one for investigating the enteropathogenicity of Aeromonas species . Possible reasons for the discrepancy between our study and the original report are discussed.

J Altern Complement Med, 2000 Jun, 6(3), 235 - 9
Improvement of C-reactive protein levels and body temperature of an elderly patient infected with Pseudomonas aeruginosa on treatment with Mao-bushi-saishin-to; Kamei T et al.; OBJECTIVE: To examine the effectiveness of Mao-bushi-saishin-to (Ma-Huang-Fu-Zi-Xi-Xin-Tang in Chinese medicine) (Tochimototenkaido Co . Ltd., Osaka, Japan), one of the traditional herbal medicines, against resistant bacterial infection . SETTING: The Nursing Center Himawari, Izumo, Japan DESIGN, PATIENT, AND PREPARATION: Half of the standard dose of Mao-bushi-saishin-to was prescribed for 7 days to one elderly patient with fever and positive C-reactive protein (CRP) levels suffering from drug resistant Pseudomonas aeruginosa . The daily standard dose of Mao-bushi-saishin-to is prepared from 1200 mg of dried extract obtained from three crude drugs, Ephedrae Herba (4 g), Asiasari Radix (3 g), and Aconiti Tuber (1 g) . It is certified by the Japanese Ministry of Health and Welfare . RESULTS: The patient's fever and CRP level returned to normal levels . CONCLUSIONS: In cases in which the fever does not fall in response to antibiotics for at least 3 days, half of the standard dose of Mao-bushi-saishin-to for 7 days might be worth trying to induce remission, especially for elder patients.

J Biol Chem, 2000 Sep 29, 275(39), 30064 - 8
Localization of the outer membrane subunit OprM of resistance-nodulation-cell division family multicomponent efflux pump in Pseudomonas aeruginosa; Nakajima A et al.; The outer membrane subunit OprM of the multicomponent efflux pump of Pseudomonas aeruginosa has been assumed to form a transmembrane xenobiotic exit channel across the outer membrane . We challenged this hypothesis to clarify the underlying ambiguity by manipulating the amino-terminal signal sequence of the OprM protein of the MexAB-OprM efflux pump in P . aeruginosa . {(3)H}Palmitate uptake experiments revealed that OprM is a lipoprotein . The following lines of evidence unequivocally established that the OprM protein functioned at the periplasmic space . (i) The OprM protein, in which a signal sequence including Cys-18 was replaced with that of periplasmic azurin, appeared in the periplasmic space but not in the outer membrane fraction, and the protein fully functioned as the pump subunit . (ii) The hybrid OprM containing the N-terminal transmembrane segment of the inner membrane protein, MexF, appeared exclusively in the inner membrane fraction . The hybrid protein containing 186 or 331 amino acid residues of MexF was fully active for the antibiotic extrusion, but a 42-residue protein was totally inactive . (iii) The mutant OprM, in which the N-terminal cysteine residue was replaced with another amino acid, appeared unmodified with fatty acid and was fractionated in both the periplasmic space and the inner membrane fraction but not in the outer membrane fraction . The Cys-18-modified OprM functioned for the antibiotic extrusion indistinguishably from that in the wild-type strain . We concluded, based on these results, that the OprM protein was anchored in the outer membrane via fatty acid(s) attached to the N-terminal cysteine residue and that the entire polypeptide moiety was exposed to the periplasmic space.

Clin Ther, 1999 Nov, 21(11), 1882 - 9
Pharmacokinetics and pharmacodynamics of aztreonam administered by continuous intravenous infusion; Burgess DS et al.; The pharmacodynamic parameter that appears to correlate best with a successful therapeutic outcome with beta-lactam antibiotics is the length of time the serum antibiotic concentration remains above the minimum inhibitory concentration (MIC) for the infecting pathogen . By maximizing this parameter, continuous administration of beta-lactam and related antibiotics by intravenous infusion could represent the optimal mode of drug administration . The pharmacokinetic and pharmacodynamic properties of ceftazidime administered by continuous intravenous infusion have been evaluated previously . Aztreonam is a monobactam antibiotic with similar pharmacokinetic and microbiologic activity to that of ceftazidime . This study evaluated the pharmacokinetic and pharmacodynamic characteristics of aztreonam administered as a continuous intravenous infusion in healthy volunteers against multiple clinical isolates . Five men and 3 women received 6 g of aztreonam administered by continuous intravenous infusion over 24 hours . Blood samples were collected before the infusion and at 0.5, 1 through 8, 12, 18, and 24 hours after the start of the infusion . Pharmacokinetic parameters were determined by standard equations . In vitro susceptibility testing was performed using National Committee for Clinical Laboratory Standards guidelines for 4 clinical isolates of gram-negative bacteria (2 each of Escherichia coli and Pseudomonas aeruginosa) . Serum inhibitory titers (SITs) were determined in duplicate for each clinical isolate at 0 and 24 hours . The subjects' mean (+/- SD) age was 29.3+/-4.4 years; mean weight, 74.6+/-14.0 kg; and calculated mean creatinine clearance, 107+/-13 mL/min . For the pharmacokinetic parameters, mean (+/- SD) values were as follows: steady-state serum concentration, 40.9+/-8.8 microg/L; half-life, 1.5+/-0.4 hours; elimination rate constant, 0.50+/-0.13 hours(-1); steady-state volume of distribution, 0.18+/-0.04 L/kg; and total body clearance, 6.1+/-1.2 L/h . The MICs were 0.0625 and 0.125 microg/mL against the 2 E coli isolates and 4 microg/mL against both P aeruginosa isolates . The median SITs against the E . coli isolates were 1:256 and 1:512, and against the P . aeruginosa isolates were 1:8 and 1:16 . At steady state, II subjects had serum concentrations of aztreonam > or =4 times the MIC for each organism . These findings suggest that further clinical study of the administration of aztreonam by continuous intravenous infusion is warranted.

Infection, 1999, 27(4-5), 268 - 71
Optimal tobramycin dosage in patients with cystic fibrosis--evidence for predictability based on previous drug monitoring; Bartel K et al.; A retrospective analysis of files of patients with cystic fibrosis and pulmonary exacerbations was performed to investigate whether an individual dosage of tobramycin once established by serum level determination allows a reliable prediction of the adequate dosage in a consecutive exacerbation . All patients hospitalized > or = 2 times between May 1997 and September 1998 with pulmonary exacerbation due to Pseudomonas aeruginosa infection susceptible to tobramycin were included.The initial dosage to tobramycin was 5 mg/kg body weight every 12 h followed by drug level determinations to establish the optimal dose . In a consecutive exacerbation the same dosage per kg body weight was used again and drug level determinations were repeated . Sixteen patients (six female = 38%) with a mean age of 24 years (median: 26 years, range: 9-33) were hospitalized for 49 pulmonary exacerbations (2-6 per patient, mean: 3, median: 2.5) . During the first episode of tobramycin treatment in the study period all trough levels were < 2 microg/ml (median: 0.6) and the peak levels were 7.1-16.9 microg/ml (median: 11.9) . In four patients the peak level was > 12 microg/ml . In 28 consecutive episodes the dosage of tobra myci n was chosen based on optimal results of previous drug level monitoring and in 27 instances (96%) the previously established optimal dose was confirmed . In five consecutive episodes the tobramycin dosage had been increased erroneously and this resulted in abnormally high peak levels in three cases . These findings suggest that a safe and therapeutic tobramycin dosage in an individual patient with cystic fibrosis is predictable based on a previously established optimal dosage.

Infection, 1999, 27 Suppl 2, S24 - 8
Combination therapy as a tool to prevent emergence of bacterial resistance; Mouton JW; Emergence of resistance is an ever increasing problem . One of the methods by which emergence of resistance may possibly be prevented, or at least delayed, is the use of combination therapy . Since the emergence of resistant mutants is a direct result of selective pressure by antimicrobial therapy, the chance of mutants resistant to two antimicrobials in the parent population being present is a product of mutation frequencies, provided that resistance mechanisms are independent . Comparative studies in in vitro pharmacokinetic models and in vivo indicate that emergence of resistance is less common when combination therapy is used . This is particularly true for microorganisms known to develop resistance relatively quickly, such as Pseudomonas aeruginosa, and resistance mechanisms which occur at a relatively high frequency.

J Otolaryngol, 2000 Jun, 29(3), 148 - 53
Clinicomicrobiologic evaluation of active tubotympanic type chronic suppurative otitis media; Khanna V et al.; OBJECTIVES: This prospective study was conducted to determine the spectrum of micro-organisms encountered in patients with active-stage chronic suppurative otitis media (CSOM) (tubotympanic type) and to see whether prescribing an antibiotic after culture sensitivity was more beneficial as compared to initial treatment without cultures . DESIGN: Prospective randomized study of 110 patients of active CSOM (tubotympanic type) divided into two groups of 55 cases each . SETTING: Departments of Ear, Nose and Throat and Microbiology of a tertiary care hospital . METHODS: The patients in group A were prescribed an antibiotic according to the culture and sensitivity, whereas in group B, culture was not done at the first visit, and a broad-spectrum antimicrobial, namely, co-trimoxazole, was prescribed blindly for a maximum period of 2 weeks . The cases that still had ear discharge were then subjected to culture and sensitivity and the antibiotic was prescribed accordingly . MAIN OUTCOME MEASURES: All patients in group A were subjected to bacterial culture and sensitivity and fungal culture . Only failed cases in group B were subjected to the same . RESULTS: In group A, 47 patients (85.50%) had positive bacterial culture and 20 patients had positive fungal culture . Pseudomonas aeruginosa was the most common bacterial isolate . All of these 47 patients had a dry ear with a maximum 2 weeks of antibiotic therapy . Among the remaining 8 patients who had negative bacterial culture, 5 patients (9.0%) showed fungal isolates on culture and responded to topical antifungal treatment . The remaining 3 failed cases (5.5%) responded to daily dry mopping alone . In group B, 41 patients (74.54%) attained a dry ear . Bacterial culture and sensitivity were done in the remaining 14 (25.46%) failed cases . The culture was positive in 11 patients (20.0%) and sterile in 3 patients (5.5%) . In the latter group, only 1 patient had fungus on culture and the remaining 2 patients responded to daily dry mopping alone, which was done at a maximum for a week only . The most common fungal pathogen isolated was Aspergillus flavus . CONCLUSIONS: Pseudomonas aeruginosa was the most common bacteria and Aspergillus flavus the most common fungus isolated in this study . In group A patients, the failed cases were less as compared to the control group B, but the p value was .2 . Hence, there is no definite role of culture and sensitivity in the initial management plan of all cases of CSOM . Ideally, every such case should be prescribed a broad-spectrum antibiotic and only in failed cases should culture and sensitivity be done.

J Antimicrob Chemother, 2000 Jul, 46(1), 133 - 6
Antibiotic susceptibility and mechanisms of beta-lactam resistance in 1310 strains of pseudomonas aeruginosa: a French multicentre study (1996); Cavallo JD et al.; A total of 1310 consecutive strains of Pseudomonas aeruginosa were collected in 11 French hospitals in 1996 . The percentages of susceptible isolates measured by the agar dilution method were: ticarcillin (53%), piperacillin (69%) (MIC 16 mg/L), ceftazidime (77%), cefepime (55%), cefpirome (40%), aztreonam (57.5%), imipenem (81.5%) (MIC 4 mg/L), amikacin (64.5%) (MIC 8 mg/L) and ciprofloxacin (58%) (MIC 1 mg/L) . Resistance to beta-lactams was linked to the production of transferable beta-lactamases (30%), overproduction of cephalosporinase (29%) and to non-enzymic mechanisms (38%).

Electrophoresis, 2000 Jun, 21(10), 1985 - 91
Separation of closely related peptide substrates of human proteinases by micellar electrokinetic chromatography with anionic and nonionic surfactants; Lupi A et al.; In order to use micellar electrokinetic chromatography to determine the proteolytic activity of different proteinases simultaneously present in physiological fluids, the technique must be able to separate mixtures of substrates with closely related structures . In an attempt to determine the best electrophoretic conditions for resolving six p-nitroanilide peptides used as synthetic substrates of the elastolytic enzymes (human neutrophil elastase, cathepsin G, Pseudomonas aeruginosa elastase) most commonly involved in pulmonary diseases, we investigated the efficiency of ionic and nonionic surfactants in achieving the separation of this complex mixture . The results presented here show that, of all the electrophoretic systems tested, 30 mM sodium tetraborate, pH 9.3, containing 25 mM Brij 35 as micellar agent offered the best performance; the separation efficiency of peptides is greater than that obtained with other reagents and all peaks are baseline resolved and unambiguously identifiable . Analysis of the micelle-solute interaction with the surfactants investigated allowed better definition of the mechanism involved in the distribution of these peptides to the micelles and identification of some structural features that determined the magnitude of the micelle peptide complex formation.

Cutis, 2000 Jun, 65(6), 359 - 62
Localized whirlpool folliculitis in a football player; Green JJ; Pseudomonas aeruginosa folliculitis occurs in patients exposed to contaminated water . Most out-breaks are associated with whirlpools . The infection is characterized by follicular, erythematous papules and pustules located on immersed body surfaces . Most reported cases are the result of recreational water use, occur in a diffuse pattern, and are devoid of green pustular pigment changes . The case described occurred in a football player after whirlpool treatment for an ankle strain . Green pustules and a localized affected area are unusual aspects of this case.

Microbiology, 2000 Jul, 146 ( Pt 7), 1717 - 25
Carbohydrate sulfation effects on growth of Pseudomonas aeruginosa; Chance DL et al.; Pseudomonas aeruginosa is a key player in the pathology and morbidity of cystic fibrosis . Chronic obstructive pulmonary disease, which results from the most common and severe mutations in this genetic disorder, typically includes chronic infection with P . aeruginosa which, even with rugged antibiotic and physical therapy regimens, is rarely eradicated . It is not known whether the increased oligosaccharide sulfation characteristic of cystic fibrosis tracheobronchial mucins plays a role in the survival of P . aeruginosa in the airway . In this study, sulfated monosaccharides were synthesized and tested for their effects on the growth of clinical isolates and laboratory strains of this organism when supplied as the sole carbon source in vitro . Carbohydrate sulfation was observed to reduce, but not prohibit, growth of P . aeruginosa on carbohydrates normally utilized in their nonsulfated form . The various sulfated sugars employed as the sole carbon source gave characteristic and consistent growth profiles and maximum growth values across the strains tested . P . aeruginosa isolates from patients with cystic fibrosis often express a mucoid phenotype, which is thought to contribute to their ability to survive in harsh conditions . Carbohydrate sulfation effects on growth did not differ significantly between mucoid and nonmucoid strains . These results suggest that the additional sulfation of tracheobronchial mucin documented in cystic fibrosis may in fact contribute to the mucin's resistance to utilization by P . aeruginosa and potentially other pathogens, providing an additional level of host protection, and limiting the available nutrient pool and thereby bacterial growth.

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